Benzimidazole thiophene compounds

ABSTRACT

The present invention provides benzimidazole thiophene compounds pharmaceutical compositions containing the same, processes for preparing the same and their use as pharmaceutical agents.

This Application claims priority to U.S. Provisional Patent ApplicationSer. Nos. 60/714,337, filed 6 Sep. 2005 and 60/786,244, filed 27 Mar.2006.

BACKGROUND OF THE INVENTION

The present invention relates to novel benzimidazole thiophenecompounds, pharmaceutical formulations comprising these compounds, andthe use of these compounds in therapy.

Polo-like kinases (“PLK”) are evolutionarily conserved serine/threoninekinases that play critical roles in regulating processes in the cellcycle. PLK plays a role in the entry into and the exit from mitosis indiverse organisms from yeast to mammalian cells. PLK includes PLK1,PLK2, PLK3 and PLK4.

Overexpression of PLK1 appears to be strongly associated with neoplasticcells (including cancers). A published study has shown high levels ofPLK1 RNA expression in >80% of lung and breast tumors, with little to noexpression in adjacent normal tissue. Several studies have showncorrelations between PLK expression, histological grade, and prognosisin several types of cancer. Significant correlations were found betweenpercentages of PLK-positive cells and histological grade of ovarian andendometrial cancer (P<0.001). These studies noted that PLK is stronglyexpressed in invading endometrial carcinoma cells and that this couldreflect the degree of malignancy and proliferation in endometrialcarcinoma. Using RT-PCR analysis, PLK overexpression was detected in 97%of esophageal carcinomas and 73% of gastric carcinomas as compared tothe corresponding normal tissues. Further, patients with high levels ofPLK overexpression in esophageal carcinoma represented a significantlypoorer prognosis group than those with low levels of PLK overexpression.In head and neck cancers, elevated mRNA expression of PLK1 was observedin most tumors; a Kaplan-Meier analysis showed that those patients withmoderate levels of PLK1 expression survived longer than those with highlevels of PLK1 expression. Analysis of patients with non-small cell lungcarcinoma showed similar outcomes related to PLK1 expression.

PCT Publication No. WO2004/014899 to SmithKline Beecham discloses novelbenzimidazole thiophene compounds of formula (I):

wherein:

-   R¹ is selected from the group consisting of H, alkyl, alkenyl,    alkynyl, —C(O)R⁷, —CO₂R⁷, —C(O)NR⁷R⁸, —C(O)N(R⁷)OR⁸,    —C(O)N(R⁷)—R²—OR⁸, —C(O)N(R⁷)-Ph, —C(O)N(R⁷)—R²-Ph,    —C(O)N(R⁷)C(O)R⁸, —C(O)N(R⁷)CO₂R⁸, —C(O)N(R⁷)C(O)NR⁷R⁸,    —C(O)N(R⁷)S(O)₂R⁸, —R²—OR⁷, —R²—O—C(O)R⁷, —C(S)R⁷, —C(S)NR⁷R⁸,    —C(S)N(R⁷)-Ph, —C(S)N(R⁷)—R²-Ph, —R²—SR⁷, —C(═NR⁷)NR⁷R⁸,    —C(═NR⁷)N(R⁸)-Ph, —C(═NR⁷)N(R⁸)—R²-Ph, —R²—NR⁷R⁸, —CN, —OR⁷,    —S(O)_(f)R⁷, —S(O)₂NR⁷R⁸, —S(O)₂N(R⁷)-Ph, —S(O)₂N(R⁷)—R²-Ph, —NR⁷R⁸,    N(R⁷)-Ph, —N(R⁷)—R²-Ph, —N(R⁷)—SO₂R⁸ and Het;-   Ph is phenyl optionally substituted from 1 to 3 times with a    substituent selected from the group consisting of halo, alkyl, —OH,    —R²—OH, —O-alkyl, —R²—O-alkyl, —NH₂, —N(H)alkyl, —N(alkyl)₂, —CN and    —N₃;-   Het is a 5-7 membered heterocycle having 1, 2, 3 or 4 heteroatoms    selected from N, O and S, or a 5-6 membered heteroaryl having 1, 2,    3 or 4 heteroatoms selected from N, O and S, each optionally    substituted from 1 to 2 times with a substituent selected from the    group consisting of halo, alkyl, oxo, —OH, —R²—OH, —O-alkyl,    —R²—O-alkyl, —NH₂, —N(H)alkyl, —N(alkyl)₂, —CN and —N₃;-   Q¹ is a group of formula: —(R²)_(a)—(Y¹)_(b)—(R²)_(c)—R³-   a, b and c are the same or different and are each independently 0 or    1 and at least one of a or b is 1;-   n is 0, 1, 2, 3 or 4;-   Q² is a group of formula: —(R²)_(aa)—(Y²)_(bb)—(R²)_(cc)—R⁴ or two    adjacent Q² groups are selected from the group consisting of alkyl,    alkenyl, —OR⁷, —S(O)_(f)R⁷ and —NR⁷R⁸ and together with the carbon    atoms to which they are bound, they form a C₅₋₆cycloalkyl,    C₅₋₆cycloalkenyl, phenyl, 5-7 membered heterocycle having 1 or 2    heteroatoms selected from N, O and S, or 5-6 membered heteroaryl    having 1 or 2 heteroatoms selected from N, O and S;-   aa, bb and cc are the same or different and are each independently 0    or 1;-   each Y¹ and Y² is the same or different and is independently    selected from the group consisting of —O—, —S(O)_(f)—, —N(R⁷)—,    —C(O)—, —OC(O)—, —CO₂—, —C(O)N(R⁷)—, —C(O)N(R⁷)S(O)₂—, —OC(O)N(R⁷)—,    —OS(O)₂—, —S(O)₂N(R⁷)—, —S(O)₂N(R⁷)C(O)—, —N(R⁷)S(O)₂—, —N(R⁷)C(O)—,    —N(R⁷)CO₂— and —N(R⁷)C(O)N(R⁷)—;-   each R² is the same or different and is independently selected from    the group consisting of alkylene, alkenylene and alkynylene;-   each R³ and R⁴ is the same or different and is each independently    selected from the group consisting of H, halo, alkyl, alkenyl,    alkynyl, —C(O)R⁷, —C(O)NR⁷R⁸, —CO₂R⁷, —C(S)R⁷, —C(S)NR⁷R⁸,    —C(═NR⁷)R⁸, —C(═NR⁷)NR⁷R⁸, —CR⁷═N—OR⁷, —OR⁷, —S(O)_(f)R⁷,    —S(O)₂NR⁷R⁸, —NR⁷R⁸, —N(R⁷)C(O)R⁸, —N(R⁷)S(O)₂R⁸, —NO₂, —CN, —N₃ and    a group of formula (ii):

-   -   wherein:    -   Ring A is selected from the group consisting of C₅₋₁₀cycloalkyl,        C₅₋₁₀cycyloalkenyl, aryl, 5-10 membered heterocycle having 1, 2        or 3 heteroatoms selected from N, O and S and 5-10 membered        heteroaryl having 1, 2 or 3 heteroatoms selected from N, O and S    -   each d is 0 or 1;    -   e is 0, 1, 2, 3 or 4;    -   each R⁶ is the same or different and is independently selected        from the group consisting of H, halo, alkyl, alkenyl, alkynyl,        cycloalkyl, cycloalkenyl, Ph, Het, —CH(OH)—R²—OH, —C(O)R⁷,        —CO₂R⁷, —CO₂—R₂-Ph, —CO₂—R²-Het, —C(O)NR⁷R⁸, —C(O)N(R⁷)C(O)R⁷,        —C(O)N(R⁷)CO₂R⁷, —C(O)N(R⁷)C(O)NR⁷R⁸, —C(O)N(R⁷)S(O)₂R⁷,        —C(S)R⁷, —C(S)NR⁷R⁸, —C(═NR⁷)R⁸, —C(═NR⁷)NR⁷R⁸, —CR⁷═N—OR⁸, ═O,        —OR⁷, —OC(O)R⁷, —OC(O) Ph, —OC(O) Het, —OC(O)NR⁷R⁸,        —O—R²—S(O)₂R⁷, —S(O)_(f)R⁷, —S(O)₂NR⁷R⁸, —S(O)₂Ph, —S(O)₂Het,        —NR⁷R⁸, —N(R⁷)C(O)R⁸, —N(R⁷)CO₂R⁸, —N(R⁷)—R²—CO₂R⁸,        —N(R⁷)C(O)NR⁷R⁸, —N(R⁷)—R²—C(O)NR⁷R⁸, —N(R⁷)C(O)Ph,        —N(R⁷)C(O)Het, —N(R⁷)Ph, —N(R⁷)Het, —N(R⁷)C(O)NR⁷—R²—NR⁷R⁸,        —N(R⁷)C(O)N(R⁷)Ph, —N(R⁷)C(O)N(R⁷)Het, —N(R⁷)C(O)N(R⁷)—R²-Het,        —N(R⁷)S(O)₂R⁸, —N(R⁷)—R²—S(O)₂R⁸, —NO₂, —CN and —N₃;

-   wherein when Q¹ is defined where b is 1 and c is 0, R³ is not halo,    —C(O)R⁷, —C(O)NR⁷R⁸, —CO₂R⁷, —C(S)R⁷, —C(S)NR⁷R⁸, —C(═NR⁷)R⁸,    —C(═NR⁷)NR⁷R⁸, —CR⁷═N—OR⁷, —OR⁷, —S(O)_(f)R⁷, —S(O)₂NR⁷R⁸, —NR⁷R⁸,    —N(R⁷)C(O)R⁸, —N(R⁷)S(O)₂R⁸, —NO₂, —CN or —N₃;

-   wherein when Q² is defined where bb is 1 and cc is 0, R⁴ is not    halo, —C(O)R⁷, —C(O)NR⁷R⁸, —CO₂R⁷, —C(S)R⁷, —C(S)NR⁷R⁸, —C(═NR⁷)R⁸,    —C(═NR⁷)NR⁷R⁸, —CR⁷═N—OR⁷, —OR⁷, —S(O)_(f)R⁷, —S(O)₂NR⁷R⁸, —NR⁷R⁸,    —N(R⁷)C(O)R⁸, —N(R⁷)S(O)₂R⁸, —NO₂, —CN or —N₃;

-   R⁵ is selected from the group consisting of H, halo, alkyl,    cycloalkyl, OR⁷, —S(O)_(f)R⁷, —NR⁷R⁸, —NHC(O)R⁷, —NHC(O)NR⁷R⁸ and    —NHS(O)₂R⁷;

-   f is 0, 1 or 2; and

-   each R⁷ and each R⁸ are the same or different and are each    independently selected from the group consisting of H, alkyl,    alkenyl, alkynyl, cycloalkyl and cycloalkenyl;

-   wherein when R¹ is —CO₂CH₃ and n is 0, Q¹ is not —OH;    or a pharmaceutically acceptable salt, solvate or physiologically    functional derivative thereof.

Also disclosed are pharmaceutical compositions containing thesecompounds, processes for their preparation and methods for treatment ofconditions mediated by PLK using these compounds.

BRIEF SUMMARY OF THE INVENTION

According to a first aspect of the invention there is provided acompound selected from:

-   -   wherein * indicates chiral carbon;        and pharmaceutically acceptable salts or solvates thereof.

In another aspect, there is provided an enantiomerically enrichedcompound selected from A, B, C, D, E, F, G, and H wherein thestereochemistry of the chiral carbon is R.

In a third aspect of the invention there is provided a pharmaceuticalcomposition comprising a compound selected from A, B, C, D, E, F, G andH and pharmaceutically acceptable salts or solvates thereof. In oneembodiment, the pharmaceutical composition further comprises apharmaceutically acceptable carrier, diluent or excipient.

In a fourth aspect of the invention, there is provided a method fortreating a condition mediated by PLK in a mammal in need thereof. Themethod comprises administering to the mammal a therapeutically effectiveamount of a compound selected from A, B, C, D, E, F, G and H andpharmaceutically acceptable salts or solvates thereof.

In a fifth aspect of the invention, there is provided a method fortreating a susceptible neoplasm in a mammal in need thereof. The methodcomprises administering to the mammal a therapeutically effective amountof a compound selected from A, B, C, D, E, F, G and H andpharmaceutically acceptable salts or solvates thereof. The susceptibleneoplasm may be selected from the group consisting of breast cancer,colon cancer, lung cancer, including small cell lung cancer andnon-small cell lung cancer, prostate cancer, endometrial cancer, gastriccancer, melanoma, ovarian cancer, pancreatic cancer, squamous cellcarcinoma, carcinoma of the head and neck, esophageal carcinoma,hepatocellular carcinoma, and hematologic malignancies, such as acuteleukemias and aggressive lymphomas.

In a sixth aspect of the invention, there is provided a method fortreating a breast cancer in a mammal in need thereof. The methodcomprises administering to the mammal a therapeutically effective amountof a compound selected from A, B, C, D, E, F, G and H andpharmaceutically acceptable salts or solvates thereof.

In a seventh aspect of the invention, there is provided a method fortreating ovarian cancer in a mammal in need thereof. The methodcomprises administering to the mammal a therapeutically effective amountof a compound selected from A, B, C, D, E, F, G and H andpharmaceutically acceptable salts or solvates thereof.

In a eighth aspect of the invention, there is provided a method fortreating non-small cell lung cancer in a mammal in need thereof. Themethod comprises administering to the mammal a therapeutically effectiveamount of a compound selected from A, B, C, D, E, F, G and H andpharmaceutically acceptable salts or solvates thereof.

In a ninth aspect of the invention, there is provided a method fortreating prostate cancer in a mammal in need thereof. The methodcomprises administering to the mammal a therapeutically effective amountof a compound selected from A, B, C, D, E, F, G and H andpharmaceutically acceptable salts or solvates thereof.

In a tenth aspect of the invention, there is provided a method fortreating a hematologic malignancy in a mammal in need thereof. Themethod comprises administering to the mammal a therapeutically effectiveamount of a compound selected from A, B, C, D, E, F, G and H andpharmaceutically acceptable salts or solvates thereof.

In another aspect of the invention, there is provided a method fortreating a condition characterized by inappropriate cellularproliferation. The method comprises contacting the cell with atherapeutically effective amount of a compound selected from A, B, C, D,E, F, G and H and pharmaceutically acceptable salts or solvates thereof.

In another aspect, the present invention provides a method forinhibiting proliferation of a cell. The method comprises contacting thecell with an amount of a compound selected from A, B, C, D, E, F, G andH and pharmaceutically acceptable salts or solvates thereof.

In another aspect, the present invention provides a method forinhibiting mitosis in a cell. The method comprises administering to thecell an amount of a compound selected from A, B, C, D, E, F, G and H andpharmaceutically acceptable salts or solvates thereof.

In another aspect, the present invention provides a compound selectedfrom A, B, C, D, E, F, G and H and pharmaceutically acceptable salts orsolvates thereof for use in therapy.

In yet another aspect, the present invention provides a compoundselected from A, B, C, D, E, F, G and H and pharmaceutically acceptablesalts or solvates thereof for use in the treatment of a conditionmediated by PLK in a mammal in need thereof.

In yet another aspect, the present invention provides a compoundselected from A, B, C, D, E, F, G and H and pharmaceutically acceptablesalts or solvates thereof for use in the treatment of a susceptibleneoplasm in a mammal.

In yet another aspect, the present invention provides a compoundselected from A, B, C, D, E, F, G and H and pharmaceutically acceptablesalts or solvates thereof for use in the treatment of breast cancer,ovarian cancer, non-small cell lung cancer, prostate cancer, or ahematologic malignancy in a mammal.

In another aspect, the present invention provides a compound selectedfrom A, B, C, D, E, F, G and H and pharmaceutically acceptable salts orsolvates thereof for use in the treatment of a condition characterizedby inappropriate cellular proliferation.

In yet another aspect, the present invention provides a compoundselected from A, B, C, D, E, F, G and H and pharmaceutically acceptablesalts or solvates thereof for use in inhibiting proliferation of a cell.

In yet another aspect, the present invention provides a compoundselected from A, B, C, D, E, F, G and H and pharmaceutically acceptablesalts or solvates thereof for use in inhibiting mitosis in a cell.

In yet another aspect, the present invention provides the use of acompound selected from A, B, C, D, E, F, G and H and pharmaceuticallyacceptable salts or solvates thereof for the preparation of a medicamentfor the treatment of condition mediated by PLK in a mammal.

In yet another aspect, the present invention provides the use of acompound selected from A, B, C, D, E, F, G and H and pharmaceuticallyacceptable salts or solvates thereof for the preparation of a medicamentfor the treatment of a susceptible neoplasm in a mammal.

In yet another aspect, the present invention provides the use of acompound selected from A, B, C, D, E, F, G and H and pharmaceuticallyacceptable salts or solvates thereof for the preparation of a medicamentfor the treatment of a breast cancer, ovarian cancer, non-small celllung cancer, prostate cancer, or a hematologic malignancy in a mammal.

In yet another aspect, the present invention provides the use of acompound selected from A, B, C, D, E, F, G and H and pharmaceuticallyacceptable salts or solvates thereof for the preparation of a medicamentfor the treatment of a condition characterized by inappropriate cellularproliferation in a mammal.

In yet another aspect, the present invention provides the use of acompound selected from A, B, C, D, E, F, G and H and pharmaceuticallyacceptable salts or solvates thereof for the preparation of a medicamentfor inhibiting proliferation of a cell.

In yet another aspect, the present invention provides the use of acompound selected from A, B, C, D, E, F, G and H and pharmaceuticallyacceptable salts or solvates thereof for the preparation of a medicamentfor inhibiting mitosis in a cell.

In yet another aspect, the present invention provides a pharmaceuticalcomposition comprising a compound selected from A, B, C, D, E, F, G andH and pharmaceutically acceptable salts or solvates thereof for use inthe treatment of a susceptible neoplasm, such as breast cancer, ovariancancer, non-small cell lung cancer, prostate cancer, and hematologicmalignancies, in a mammal.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “compound(s) of the invention” or “compound(s) selectedfrom A, B, C, D, E, F, G and H” means compound(s) selected from A, B, C,D, E, F, G and H, or enantiomerically enriched compound(s) selected fromA, B, C, D, E, F, G and H, or a pharmaceutically acceptable salt orsolvate thereof.

As used herein, “compound(s) selected from A-1, B-1, C-1, D-1, E-1, F-1,G-1 and H-1” means compound(s) selected from A-1, B-1, C-1, D-1, E-1,F-1, G-1 and H-1 or a pharmaceutically acceptable salt or solvatethereof.

As used herein, the term “optionally” means that the subsequentlydescribed event(s) may or may not occur, and includes both event(s) thatoccur and events that do not occur.

Compounds of the invention exist in stereoisomeric forms (e.g. theycontain one or more chiral or asymmetric carbon atoms). The term“chiral” refers to a molecule that is not superimposable on its mirrorimage. The term “achiral” refers to a molecule that is superimposable onits mirror image.

The term “stereoisomers” refers to compounds which are have a commonchemical constitution but differ in the arrangement of the atoms orgroups in space. Stereoisomers may be optical isomers or geometricisomers. Optical isomers include both enantiomers and diastereomers. An“enantiomer” is one of a pair of optical isomers containing a chiralcarbon atom whose molecular configuration have left- and right-hand(chiral) forms. That is, “enantiomer” refers to each of a pair ofoptical isomers of a compound which are non-superimposable mirror imagesof one another. A “diastereomer” is one of a pair of optical isomers ofa compound with two or more centers of dissymmetry and whose moleculesare not mirror images of one another. The nomenclature of a chiralcenter is governed by the (R)—(S) system. Whether a particular compoundis designated as the “R” or “S” enantiomer according to the systemdepends upon the nature of the atoms or groups which are bound to thechiral carbon.

Enantiomers differ in their behavior toward plane-polarized light, thatis, their optical activity. An enantiomer that rotates plane-polarizedlight in a clockwise direction is said to be dextrorotatory and isdesignated by the symbol “d” or “(+)” for positive rotation. Anenantiomer that rotates plane-polarized light in the counterclockwisedirection is said to be levorotatory and is designated by the symbol “I”or “(−)” for negative rotation. There is no correlation between theconfiguration of enantiomers and the direction in which they rotateplane-polarized light. There is also no necessary correlation betweenthe (R) and (S) designation and the direction of rotation of theplane-polarized light. The optical activity, or direction of rotation ofplane-polarized light, of an enantiomer of a compound of the inventionmay be determined using conventional techniques.

The terms “racemate” and “racemic mixture” as used herein refer to amixture of the (R)- and the (S)-optical isomers (e.g., enantiomers) of acompound in equal, i.e. 50:50 proportion.

The term “enantiomerically enriched” as used herein refers topreparations comprising a mixture of optical isomers in which thequantity of one enantiomer is higher than the quantity of the other.Thus, “enantiomerically enriched” refers to mixtures of optical isomerswherein the ratio of enantiomer is greater than 50:50. Anenantiomerically enriched compound comprises greater than 50% by weightof one enantiomer relative to the other. For example enantiomericallyenriched5-{6-[(Methylsulfonyl)methyl]-1H-benzimidazol-1-yl}-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxamiderefers to a composition comprising greater than 50% by weight of the(R)-enantiomer relative to the (S)-enantiomer of the compound. In oneembodiment, an enantiomerically enriched compound comprises at least 75%by weight of one enantiomer relative to the other. In anotherembodiment, an enantiomerically enriched compound comprises at least 80%by weight of one enantiomer relative to the other. In one particularembodiment, an enantiomerically enriched compound comprises at least 85%by weight of one enantiomer relative to the other.

The term “enantiomerically pure” as used herein refers toenantiomerically enriched compounds comprising at least 90% by weight ofone enantiomer relative to the other. In one embodiment, anenantiomerically pure compound comprises at least 95% by weight of oneenantiomer relative to the other. In one particular embodiment, anenantiomerically pure compound comprises at least 99% by weight of oneenantiomer relative to the other.

The present invention provides compounds selected from:

-   -   wherein * indicates chiral carbon;        and pharmaceutically acceptable salts and solvates thereof.

In one particular embodiment, the compounds A, B, C, D, E, F, G and Hare enantiomerically enriched, wherein the stereochemistry of the chiralcarbon is R. In another embodiment the compounds A, B, C, D, E, F, G andH are enantiomerically pure, wherein the stereochemistry of the chiralcarbon is R.

Thus, in one preferred embodiment, the present invention providesenantionmerically enriched and enantiomerically pure compounds selectedfrom:

-   5-{6-[(Methylsulfonyl)methyl]-1H-benzimidazol-1-yl}-3-({(1R)-1-[2-(trifluoromethyl)phenyl]-ethyl}oxy)-2-thiophenecarboxamide;

-   3-{[(1R)-1-(2-Chlorophenyl)ethyl]oxy}-5-{6-[(methylsulfonyl)methyl]-1H-benzimidazol-1-yl}-2-thiophenecarboxamide;

-   5-{6-[(4-Methylpiperazin-1-yl)methyl]-1H-benzimidazol-1-yl}-3-{(1R)-1-[2-(trifluoromethyl)phenyl]ethoxy}thiophene-2-carboxamide;

-   3-{[(1R)-1-(2-Chlorophenyl)ethyl]oxy}-5-{6-[(4-methylpiperazin-1-yl)methyl]-1H-benzimidazol-1-yl}thiophene-2-carboxamide;

-   5-[6-(4-Piperidinyloxy)-1H-benzimidazol-1-yl]-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxamide;

-   3-{[(1R)-1-(2-Chlorophenyl)ethyl]oxy}-5-[6-(4-piperidinyloxy)-1H-benzimidazol-1-yl]-2-thiophenecarboxamide;

-   5-{6-[(1-Methyl-4-piperidinyl)oxy]-1H-benzimidazol-1-yl}-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxamide;    and

-   3-{[(1R)-1-(2-Chlorophenyl)ethyl]oxy}-5-{6-[(1-methyl-4-piperidinyl)oxy]-1H-benzimidazol-1-yl}-2-thiophenecarboxamide;    and pharmaceutically acceptable salts and solvates thereof.

It will be appreciated by those skilled in the art that the compounds ofthe present invention may be utilized in the form of a pharmaceuticallyacceptable salt or solvate thereof. The pharmaceutically acceptablesalts of the compounds of the present invention (or the enantiomericallyenriched or pure forms thereof) include conventional salts formed frompharmaceutically acceptable inorganic or organic acids or bases as wellas quaternary ammonium salts. More specific examples of suitable acidsalts include hydrochloric, hydrobromic, sulfuric, phosphoric, nitric,perchloric, fumaric, acetic, propionic, succinic, glycolic, formic,lactic, maleic, tartaric, citric, palmoic, malonic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicylic, fumaric, toluenesulfonic,methanesulfonic (mesylate), naphthalene-2-sulfonic, benzenesulfonichydroxynaphthoic, hydroiodic, malic, steroic, tannic and the like. Otheracids such as oxalic, while not in themselves pharmaceuticallyacceptable, may be useful in the preparation of salts useful asintermediates in obtaining the compounds of the invention and theirpharmaceutically acceptable salts. Specific examples of suitable basicsalts include sodium, lithium, potassium, magnesium, aluminium, calcium,zinc, N,N′-dibenzylethylenediamine, chloroprocaine, choline,diethanolamine, ethylenediamine, N-methylglucamine and procaine salts.

The term “solvate” as used herein refers to a complex of variablestoichiometry formed by a solute (a compound of the invention or anenaniomerically enriched or pure form thereof) and a solvent. Solvents,by way of example, include water, methanol, ethanol, or acetic acid.

Processes for preparing pharmaceutically acceptable salts and solvatesof the compounds of the invention are conventional in the art. See,e.g., Burger's Medicinal Chemistry And Drug Discovery 5th Edition, Vol1: Principles And Practice.

As will be apparent to those skilled in the art, in the processesdescribed below for the preparation of the compounds of the invention,certain intermediates, may alternatively be in the form ofpharmaceutically acceptable salts or solvates of the compound. Thoseterms as applied to any intermediate employed in the process ofpreparing the compounds of the invention have the same meanings as notedabove with respect to the compounds of the invention. Processes forpreparing pharmaceutically acceptable salts and solvates of suchintermediates are known in the art and are analogous to the process forpreparing pharmaceutically acceptable salts and solvates of thecompounds of the invention.

The compounds of the present invention are typically inhibitors of PLK.By PLK inhibitor is meant a compound which exhibits pIC₅₀ greater than 6in the PLK Inhibition assay described below in the examples or an IC₅₀less than 10 μM in the Methylene Blue or Cell-Titre Glo GrowthInhibition assay described below in the examples; more particularly aPLK inhibitor is a compound which exhibits a pIC₅₀ greater than 7 or anIC₅₀ less than 1 μM using the methods described in the examples below.

The present invention further provides compounds of the invention foruse in medical therapy in an animal, e.g. a mammal such as a human. Inparticular, the present invention provides compounds for use in thetreatment of a condition mediated by PLK. The present invention alsoprovides compounds for use in the treatment of a susceptible neoplasm.The term “susceptible neoplasm” is defined below. In particular, thepresent invention provides compounds for use in the treatment of avariety of solid tumors including but not limited to breast cancer,ovarian cancer, non-small cell lung cancer prostate cancer, andhematologic malignancies including but not limited to acute (myeloid andlymphoid) leukemias and aggressive lymphomas.

The present invention provides compounds for use in treating a conditioncharacterized by inappropriate cellular proliferation. The presentinvention also provides compounds for use in inhibiting proliferation ofa cell. The present invention also provides compounds for use ininhibiting mitosis in a cell.

The present invention provides methods for the treatment of severalconditions or diseases, all of which comprise the step of administeringa therapeutically effective amount of a compound of the invention. Asused herein, the term “treatment” refers to alleviating the specifiedcondition, eliminating or reducing the symptoms of the condition,slowing or eliminating the progression of the condition and preventingor delaying the reoccurrence of the condition in a previously afflictedsubject.

As used herein, the term “therapeutically effective amount” means anamount of a compound of the invention which is sufficient, in thesubject to which it is administered, to elicit the biological or medicalresponse of a cell culture, tissue, system, animal (including human)that is being sought, for instance, by a researcher or clinician. Forexample, a therapeutically effective amount of a compound of theinvention for the treatment of a condition mediated by PLK is an amountsufficient to treat the PLK mediated condition in the subject.Similarly, a therapeutically effective amount of a compound of theinvention for the treatment of a susceptible neoplasm is an amountsufficient to treat the susceptible neoplasm in the subject. In oneembodiment of the present invention, the therapeutically effectiveamount of a compound of the invention is an amount sufficient to inhibitcell mitosis. In one embodiment of the present invention, atherapeutically effective amount of a compound of the invention is anamount sufficient to regulate, modulate, bind or inhibit PLK.

The precise therapeutically effective amount of a compound of theinvention will depend on a number of factors including, but not limitedto, the age and weight of the subject being treated, the precisedisorder requiring treatment and its severity, the nature of theformulation, and the route of administration, and will ultimately be atthe discretion of the attendant physician or veternarian. Typically, acompound of the invention will be given for treatment in the range of0.1 to 200 mg/kg body weight of recipient (animal) per day or per doseor per cycle of treatment and more usually in the range of 1 to 100mg/kg body weight per day or per dose or per cycle of treatment.Acceptable dosages, may be from about 0.1 to about 2000 mg per day,dose, or cycle of treatment, and preferably from about 0.1 to about 500mg per day, dose, or cycle of treatment.

As one aspect, the present invention provides methods of regulating,modulating, binding, or inhibiting PLK for the treatment of conditionsmediated by PLK, particularly PLK1. “Regulating, modulating, binding orinhibiting PLK” refers to regulating, modulating, binding or inhibitingPLK, particularly PLK1, activity, as well as regulating, modulating,binding or inhibiting overexpression of PLK, particularly PLK1. Suchconditions include certain neoplasms (including cancers and tumors)which have been associated with PLK, particularly PLK1, and conditionscharacterized by inappropriate cellular proliferation.

The present invention provides a method for treating a conditionmediated by PLK, particularly PLK1, which comprises administering to theanimal a therapeutically effective amount of the compound of theinvention. This method and other methods of the present invention areuseful for the treatment of an animal such as a mammal and in particularhumans. Conditions which are mediated by PLK are known in the art andinclude but are not limited to neoplasms and conditions characterized byinappropriate cellular proliferation.

The present invention also provides a method for treating a susceptibleneoplasm (cancer or tumor) in an animal such as a mammal (e.g., a human)in need thereof, which method comprises administering to the animal atherapeutically effective amount of the compound of the invention.“Susceptible neoplasm” as used herein refers to neoplasms which aresusceptible to treatment with a PLK inhibitor. Neoplasms which have beenassociated with PLK and are therefore susceptible to treatment with aPLK inhibitor are known in the art, and include both primary andmetastatic tumors and cancers. For example, susceptible neoplasms withinthe scope of the present invention include but are not limited to breastcancer, colon cancer, lung cancer (including small cell lung cancer andnon-small cell lung cancer), prostate cancer, endometrial cancer,gastric cancer, melanoma, ovarian cancer, pancreatic cancer, squamouscell carcinoma, carcinoma of the head and neck, esophageal carcinoma,hepatocellular carcinoma, and hematologic malignancies including but notlimited to acute leukemias and aggressive lymphomas. In one particularembodiment, the present invention provides a method of treating breastcancer in an animal, such as a mammal (e.g., a human) in need thereof byadministering a therapeutically effective amount of a compound of thepresent invention. In another particular embodiment, the presentinvention provides a method of treating ovarian cancer in an animal,such as a mammal (e.g., a human) in need thereof by administering atherapeutically effective amount of a compound of the present invention.In another particular embodiment, the present invention provides amethod of treating non-small cell lung cancer in an animal, such as amammal (e.g., a human) in need thereof by administering atherapeutically effective amount of a compound of the present invention.In another particular embodiment, the present invention provides amethod of treating prostate cancer in an animal, such as a mammal (e.g.,a human) in need thereof by administering a therapeutically effectiveamount of a compound of the present invention. In another particularembodiment, the present invention provides a method of treating acuteleukemia, including acute myeloid leukemia and acute lymphoid leukemia,in an animal, such as a mammal (e.g., a human) in need thereof byadministering a therapeutically effective amount of a compound of thepresent invention. In another particular embodiment, the presentinvention provides a method of treating aggressive lymphoma in ananimal, such as a mammal (e.g., a human) in need thereof byadministering a therapeutically effective amount of a compound of thepresent invention.

The compounds of the invention can be used alone in the treatment ofsuch susceptible neoplasms or can be used to provide additive orsynergistic effects with one or more other compounds of the invention,or in combination with certain existing chemotherapies and/or otheranti-neoplastic therapies. In addition, the compounds of the inventioncan be used to restore effectiveness of one or more other compounds ofthe invention, certain existing chemotherapies and/or otheranti-neoplastic therapies. As used herein, “anti-neoplastic therapies”includes but is not limited to cytotoxic chemotherapy, hormonal therapy,targeted kinase inhibitors, therapeutic monoclonal antibodies, surgeryand radiation therapy.

The present invention also provides a method for treating a conditioncharacterized by inappropriate cellular proliferation in an animal, suchas a mammal (e.g., a human) in need thereof. The method comprisesadministering a therapeutically effective amount of a compound of thepresent invention. By “inappropriate cellular proliferation” is meantcellular proliferation resulting from inappropriate cell growth,cellular proliferation resulting from excessive cell division, cellularproliferation resulting from cell division at an accelerated rate,cellular proliferation resulting from inappropriate cell survival,and/or cellular proliferation in a normal cell occurring at a normalrate, which is nevertheless undesired. Conditions characterized byinappropriate cellular proliferation include but are not limited toneoplasms, blood vessel proliferative disorders, fibrotic disorders,mesangial cell proliferative disorders and inflammatory/immune-mediateddiseases. Blood vessel proliferative disorders include arthritis andrestenosis. Fibrotic disorders include hepatic cirrhosis andatherosclerosis. Mesangial cell proliferative disorders includeglomerulonephritis, malignant nephrosclerosis, and glomerulopathies.Inflammatory/immune-mediated disorders include psoriasis, chronic woundhealing, organ transplant rejection, thrombotic microangiopathysyndromes and neurodegenerative diseases. Osteoarthritis and otherosteoclast proliferation dependent diseases of excess bone resorbtionare examples of conditions characterized by inappropriate cellularproliferation in which the cellular proliferation occurs in normal cellsat a normal rate, but is nevertheless undesired.

The present invention also provides a method for inhibitingproliferation of a cell, which method comprises contacting the cell withan amount of a compound of the invention sufficient to inhibitproliferation of the cell. In one particular embodiment, the cell is aneoplastic cell. In one particular embodiment, the cell is aninappropriately proliferative cell. The term “inappropriatelyproliferative cell” as used herein refers to cells that growinappropriately (abnormally), cells that divide excessively or at anaccelerated rate, cells that inappropriately (abnormally) survive and/ornormal cells that proliferate at a normal rate but for whichproliferation is undesired. Neoplastic cells (including cancer cells)are an example of inappropriately proliferative cells but are not theonly inappropriately proliferative cells.

PLK is essential for cellular mitosis and accordingly, the compounds ofthe invention are believed to be effective for inhibiting mitosis.“Inhibiting mitosis” refers to inhibiting the entry into the M phase ofthe cell cycle, inhibiting the normal progression of the M phase of thecell cycle once M phase has been entered and inhibiting the normal exitfrom the M phase of the cell cycle. Thus, the compounds of the presentinvention may inhibit mitosis by inhibiting the cell's entry intomitosis, by inhibiting the cell's progression through mitosis or byinhibiting the cell's exit from mitosis. As one aspect, the presentinvention provides a method for inhibiting mitosis in a cell, whichmethod comprises administering to the cell an amount of a compound ofthe invention sufficient to inhibit mitosis. In one particularembodiment, the cell is a neoplastic cell. In one particular embodiment,the cell is an inappropriately proliferative cell.

The present invention also provides the use of a compound of theinvention for the preparation of a medicament for the treatment ofcondition mediated by PLK in an animal, such as a mammal (e.g., ahuman). The present invention further provides the use of a compound forthe preparation of a medicament for the treatment of a susceptibleneoplasm in an animal. In particular, the present invention provides theuse of a compound for the preparation of a medicament for the treatmentof a breast cancer in an animal. The present invention also provides theuse of a compound for the preparation of a medicament for the treatmentof ovarian cancer in an animal. The present invention provides the useof a compound for the preparation of a medicament for the treatment ofnon-small cell lung cancer in an animal. The present invention alsoprovides the use of a compound for the preparation of a medicament forthe treatment of prostate cancer in an animal. The present inventionprovides the use of a compound for the preparation of a medicament forthe treatment of acute leukemia (including acute myeloid and acutelymphoid leukemia) in an animal. The present invention provides the useof a compound for that the preparation of a medicatment for thetreatment of aggressive lymphoma in an animal. The present inventionfurther provides the use of a compound for the preparation of amedicament for the treatment of a condition characterized byinappropriate cellular proliferation. The present invention furtherprovides the use of a compound for the preparation of a medicament forinhibiting proliferation of a cell. The present invention furtherprovides the use of a compound for the preparation of a medicament forinhibiting mitosis in a cell.

While it is possible that, for use in therapy, a therapeuticallyeffective amount of a compound of the invention may be administered asthe raw chemical, it is typically presented as the active ingredient ofa pharmaceutical composition or formulation. Accordingly, the inventionfurther provides a pharmaceutical composition comprising a compound ofthe invention. The pharmaceutical composition may further comprise oneor more pharmaceutically acceptable carriers, diluents, and/orexcipients. The carrier(s), diluent(s) and/or excipient(s) must beacceptable in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof. Inaccordance with another aspect of the invention there is also provided aprocess for the preparation of a pharmaceutical formulation includingadmixing a compound of the invention with one or more pharmaceuticallyacceptable carriers, diluents and/or excipients.

Pharmaceutical formulations may be presented in unit dose formcontaining a predetermined amount of active ingredient per unit dose.Such a unit may contain a therapeutically effective dose of the compoundof the invention or a fraction of a therapeutically effective dose suchthat multiple unit dosage forms might be administered at a given time toachieve the desired therapeutically effective dose. Preferred unitdosage formulations are those containing a daily dose or sub-dose, asherein above recited, or an appropriate fraction thereof, of an activeingredient. Furthermore, such pharmaceutical formulations may beprepared by any of the methods well known in the pharmacy art.

Pharmaceutical formulations may be adapted for administration by anyappropriate route, for example by the oral (including buccal orsublingual), rectal, nasal, topical (including buccal, sublingual ortransdermal), vaginal or parenteral (including subcutaneous,intramuscular, intravenous or intradermal) route. Such formulations maybe prepared by any method known in the art of pharmacy, for example bybringing into association the active ingredient with the carrier(s) orexcipient(s).

Pharmaceutical formulations adapted for oral administration may bepresented as discrete units such as capsules or tablets; powders orgranules; solutions or suspensions in aqueous or non-aqueous liquids;edible foams or whips; or oil-in-water liquid emulsions or water-in-oilliquid emulsions.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Powders are prepared by comminuting thecompound to a suitable fine size and mixing with a similarly comminutedpharmaceutical carrier such as an edible carbohydrate, as, for example,starch or mannitol. Flavoring, preservative, dispersing and coloringagent can also be present.

Capsules are made by preparing a powder mixture as described above, andfilling formed gelatin sheaths. Glidants and lubricants such ascolloidal silica, talc, magnesium stearate, calcium stearate or solidpolyethylene glycol can be added to the powder mixture before thefilling operation. A disintegrating or solubilizing agent such asagar-agar, calcium carbonate or sodium carbonate can also be added toimprove the availability of the medicament when the capsule is ingested.

Moreover, when desired or necessary, suitable binders, lubricants,disintegrating agents and coloring agents can also be incorporated intothe mixture. Suitable binders include starch, gelatin, natural sugarssuch as glucose or beta-lactose, corn sweeteners, natural and syntheticgums such as acacia, tragacanth or sodium alginate,carboxymethylcellulose, polyethylene glycol, waxes and the like.Lubricants used in these dosage forms include sodium oleate, sodiumstearate, magnesium stearate, sodium benzoate, sodium acetate, sodiumchloride and the like. Disintegrators include, without limitation,starch, methyl cellulose, agar, bentonite, xanthan gum and the like.Tablets are formulated, for example, by preparing a powder mixture,granulating or slugging, adding a lubricant and disintegrant andpressing into tablets. A powder mixture is prepared by mixing thecompound, suitably comminuted, with a diluent or base as describedabove, and optionally, with a binder such as carboxymethylcellulose, analiginate, gelatin, or polyvinyl pyrrolidone, a solution retardant suchas paraffin, a resorption accelerator such as a quarternary salt and/oran absorption agent such as bentonite, kaolin or dicalcium phosphate.The powder mixture can be granulated by wetting with a binder such assyrup, starch paste, acadia mucilage or solutions of cellulosic orpolymeric materials and forcing through a screen. As an alternative togranulating, the powder mixture can be run through the tablet machineand the result is imperfectly formed slugs broken into granules. Thegranules can be lubricated to prevent sticking to the tablet formingdies by means of the addition of stearic acid, a stearate salt, talc ormineral oil. The lubricated mixture is then compressed into tablets. Thecompounds of the present invention can also be combined with a freeflowing inert carrier and compressed into tablets directly without goingthrough the granulating or slugging steps. A clear or opaque protectivecoating consisting of a sealing coat of shellac, a coating of sugar orpolymeric material and a polish coating of wax can be provided.Dyestuffs can be added to these coatings to distinguish different unitdosages.

Oral fluids such as solution, syrups and elixirs can be prepared indosage unit form so that a given quantity contains a predeterminedamount of active ingredient. Syrups can be prepared by dissolving thecompound in a suitably flavored aqueous solution, while elixirs areprepared through the use of a non-toxic alcoholic vehicle. Suspensionscan be formulated by dispersing the compound in a non-toxic vehicle.Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols andpolyoxy ethylene sorbitol ethers, preservatives, flavor additive such aspeppermint oil or natural sweeteners or saccharin or other artificialsweeteners, and the like can also be added.

Where appropriate, dosage unit formulations for oral administration canbe microencapsulated. The formulation can also be prepared to prolong orsustain the release as for example by coating or embedding particulatematerial in polymers, wax or the like.

The compounds of the invention can also be administered in the form ofliposome delivery systems, such as small unilamellar vesicles, largeunilamellar vesicles and multilamellar vesicles. Liposomes can be formedfrom a variety of phospholipids, such as cholesterol, stearylamine orphosphatidylcholines.

The compounds of the invention may also be delivered by the use ofmonoclonal antibodies as individual carriers to which the compoundmolecules are coupled. The compounds may also be coupled with solublepolymers as targetable drug carriers. Such polymers can includepeptides, polyvinylpyrrolidone, pyran copolymer,polyhydroxypropylmethacrylamide-phenol,polyhydroxyethylaspartamidephenol, or polyethyleneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compounds may becoupled to a class of biodegradable polymers useful in achievingcontrolled release of a drug, for example, polylactic acid, polepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacrylates and cross-linked or amphipathicblock copolymers of hydrogels.

Pharmaceutical formulations adapted for transdermal administration maybe presented as discrete patches intended to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time. Forexample, the active ingredient may be delivered from the patch byiontophoresis as generally described in Pharmaceutical Research,3(6):318 (1986).

Pharmaceutical formulations adapted for topical administration may beformulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, sprays, aerosols or oils.

For treatments of the eye or other external tissues, for example mouthand skin, the formulations are preferably applied as a topical ointmentor cream. When formulated in an ointment, the active ingredient may beemployed with either a paraffinic or a water-miscible ointment base.Alternatively, the active ingredient may be formulated in a cream withan oil-in-water cream base or a water-in-oil base.

Pharmaceutical formulations adapted for topical administrations to theeye include eye drops wherein the active ingredient is dissolved orsuspended in a suitable carrier, especially an aqueous solvent.

Pharmaceutical formulations adapted for topical administration in themouth include lozenges, pastilles and mouth washes.

Pharmaceutical formulations adapted for rectal administration may bepresented as suppositories or as enemas.

Pharmaceutical formulations adapted for nasal administration wherein thecarrier is a solid include a coarse powder having a particle size forexample in the range 20 to 500 microns which is administered in themanner in which snuff is taken, i.e. by rapid inhalation through thenasal passage from a container of the powder held close up to the nose.Suitable formulations wherein the carrier is a liquid, foradministration as a nasal spray or as nasal drops, include aqueous oroil solutions of the active ingredient.

Pharmaceutical formulations adapted for administration by inhalationinclude fine particle dusts or mists, which may be generated by means ofvarious types of metered, dose pressurised aerosols, nebulizers orinsufflators.

Pharmaceutical formulations adapted for vaginal administration may bepresented as pessaries, tampons, creams, gels, pastes, foams or sprayformulations.

Pharmaceutical formulations adapted for parenteral administrationinclude aqueous and non-aqueous sterile injection solutions which maycontain anti-oxidants, buffers, bacteriostats and solutes which renderthe formulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations may include other agents conventionalin the art having regard to the type of formulation in question, forexample those suitable for oral administration may include flavouringagents.

In the above-described methods of treatment and uses, a compound of theinvention may be employed alone, in combination with one or more othercompounds of the invention or in combination with other therapeuticagents and/or in combination with other anti-neoplastic therapies. Inparticular, in methods of treating conditions mediated by PLK andmethods of treating susceptible neoplasms, combination with otherchemotherapeutic agents is envisaged as well as combination withsurgical therapy and radiation therapy. The term “chemotherapeutic” asused herein refers to any chemical agent having a therapeutic effect onthe subject to which it is administered. “Chemotherapeutic” agentsinclude but are not limited to anti-neoplastic agents, analgesics andanti-emetics. As used herein, “anti-neoplastic agents” include bothcytostatic and cytotoxic agents such as but not limited to cytotoxicchemotherapy, hormonal therapy, targeted kinase inhibitors andtherapeutic monoclonal antibodies. Combination therapies according tothe present invention thus comprise the administration of at least onecompound of the invention and the use of at least one other cancertreatment method. In one embodiment, combination therapies according tothe present invention comprise the administration of at least onecompound of the invention and at least one other chemotherapeutic agent.In one particular embodiment, the present invention comprises theadministration of at least one compound of the invention and at leastone anti-neoplastic agent. As an additional aspect, the presentinvention provides the methods of treatment and uses as described above,which comprise administering a compound of the invention together withat least one chemotherapeutic agent. In one particular embodiment, thechemotherapeutic agent is an anti-neoplastic agent. In anotherembodiment, the present invention provides a pharmaceutical compositionas described above further comprising at least one otherchemotherapeutic agent, more particularly, the chemotherapeutic agent isan anti-neoplastic agent.

Typically, any chemotherapeutic agent that has activity versus asusceptible neoplasm being treated may be utilized in combination withthe compounds of the invention, provided that the particular agent isclinically compatible with therapy employing a compound of theinvention. Typical anti-neoplastic agents useful in the presentinvention include, but are not limited to, anti-microtubule agents suchas diterpenoids and vinca alkaloids; platinum coordination complexes;alkylating agents such as nitrogen mustards, oxazaphosphor-ines,alkylsulfonates, nitrosoureas, and triazenes; antibiotic agents such asanthracyclins, actinomycins and bleomycins; topoisomerase II inhibitorssuch as epipodophyllotoxins; antimetabolites such as purine andpyrimidine analogues and anti-folate compounds; topoisomerase Iinhibitors such as camptothecins; hormones and hormonal analogues;signal transduction pathway inhibitors; non-receptor tyrosine kinaseangiogenesis inhibitors; immunotherapeutic agents; proapoptotic agents;and cell cycle signaling inhibitors.

Anti-microtubule or anti-mitotic agents are phase specific agents activeagainst the microtubules of tumor cells during M or the mitosis phase ofthe cell cycle. Examples of anti-microtubule agents include, but are notlimited to, diterpenoids and vinca alkaloids. Examples of diterpenoidsinclude, but are not limited to, paclitaxel and its analog docetaxel.Examples of vinca alkaloids include, but are not limited to,vinblastine, vincristine, and vinorelbine. Platinum coordinationcomplexes are non-phase specific anti-neoplastic agents, which areinteractive with DNA. The platinum complexes enter tumor cells, undergo,aquation and form intra- and interstrand crosslinks with DNA causingadverse biological effects to the tumor. Examples of platinumcoordination complexes include, but are not limited to, oxaliplatin,cisplatin and carboplatin.

Alkylating agents are non-phase specific anti-neoplastic agents andstrong electrophiles. Typically, alkylating agents form covalentlinkages, by alkylation, to DNA through nucleophilic moieties of the DNAmolecule such as phosphate, amino, and hydroxyl groups. Such alkylationdisrupts nucleic acid function leading to cell death. Examples ofalkylating agents include, but are not limited to, nitrogen mustardssuch as cyclophosphamide, melphalan, and chlorambucil; alkyl sulfonatessuch as busulfan; nitrosoureas such as carmustine; and triazenes such asdacarbazine.

Antibiotic chemotherapeutic agents are non-phase specific agents, whichbind or intercalate with DNA. Typically, such action results in stableDNA complexes or strand breakage, which disrupts ordinary function ofthe nucleic acids leading to cell death. Examples of antibioticanti-neoplastic agents include, but are not limited to, actinomycinssuch as dactinomycin, anthracyclins such as daunorubicin anddoxorubicin; and bleomycins.

Topoisomerase II inhibitors include, but are not limited to,epipodophyllotoxins.

Epipodophyllotoxins are phase specific anti-neoplastic agents derivedfrom the mandrake plant. Epipodophyllotoxins typically affect cells inthe S and G₂ phases of the cell cycle by forming a ternary complex withtopoisomerase II and DNA causing DNA strand breaks. The strand breaksaccumulate and cell death follows. Examples of epipodophyllotoxinsinclude, but are not limited to, etoposide and teniposide.

Antimetabolite neoplastic agents are phase specific anti-neoplasticagents that act at S phase (DNA synthesis) of the cell cycle byinhibiting DNA synthesis or by inhibiting purine or pyrimidine basesynthesis and thereby limiting DNA synthesis. Consequently, S phase doesnot proceed and cell death follows. Examples of antimetaboliteanti-neoplastic agents include, but are not limited to, fluorouracil,methotrexate, cytarabine, mercaptopurine and thioguanine.

Camptothecins, including, camptothecin and camptothecin derivatives areavailable or under development as Topoisomerase I inhibitors.

Camptothecins cytotoxic activity is believed to be related to itsTopoisomerase I inhibitory activity. Examples of camptothecins include,but are not limited to irinotecan, topotecan, and the various opticalforms of7-(4-methylpiperazino-methylene)-10,11-ethylenedioxy-20-camptothecin.

Hormones and hormonal analogues are useful compounds for treatingcancers in which there is a relationship between the hormone(s) andgrowth and/or lack of growth of the cancer. Examples of hormones andhormonal analogues believed to be useful in the treatment of neoplasmsinclude, but are not limited to, adrenocorti-costeroids such asprednisone and prednisolone which are useful in the treatment ofmalignant lymphoma and acute leukemia in children; aminoglutethimide andother aromatase inhibitors such as anastrozole, letrazole, vorazole, andexemestane useful in the treatment of adrenocortical carcinoma andhormone dependent breast carcinoma containing estrogen receptors;progestrins such as megestrol acetate useful in the treatment of hormonedependent breast cancer and endometrial carcinoma; estrogens, androgens,and anti-androgens such as flutamide, nilutamide, bicalutamide,cyproterone acetate and 5α-reductases such as finasteride anddutasteride, useful in the treatment of prostatic carcinoma and benignprostatic hypertrophy; anti-estrogens such as tamoxifen, toremifene,raloxifene, droloxifene and iodoxyfene useful in the treatment ofhormone dependent breast carcinoma; and gonadotropin-releasing hormone(GnRH) and analogues thereof, such as goserelin acetate and leuprolide,which stimulate the release of leutinizing hormone (LH) and/or folliclestimulating hormone (FSH) with short-term or intermittent use but leadto suppression of LH and FSH with long-term use indicated for thetreatment prostatic carcinoma and hormone dependent breast carcinoma.

Signal transduction pathway inhibitors are those inhibitors which blockor inhibit a chemical process which evokes an intracellular change. Asused herein this change is cell proliferation, survival, angiogenesis ordifferentiation. Signal tranduction inhibitors useful in the presentinvention include inhibitors of receptor tyrosine kinases, non-receptortyrosine kinases, SH2/SH3 domain blockers, serine/threonine kinases,phosphotidyl inositol-3 kinases, myo-inositol signaling, and Rasoncogenes.

Several protein tyrosine kinases catalyse the phosphorylation ofspecific tyrosyl residues in various proteins involved in the regulationof cell growth. Such protein tyrosine kinases can be broadly classifiedas receptor or non-receptor kinases.

Receptor tyrosine kinases are transmembrane proteins having anextracellular ligand binding domain, a transmembrane domain, and atyrosine kinase domain. Receptor tyrosine kinases are involved in theregulation of cell growth and are sometimes termed growth factorreceptors. Inappropriate or uncontrolled activation of many of thesekinases, i.e. aberrant kinase growth factor receptor activity, forexample by over-expression or mutation, has been shown to result inuncontrolled cell growth. Accordingly, the aberrant activity of suchkinases has been linked to malignant tissue growth. Consequently,inhibitors of such kinases could provide cancer treatment methods.Growth factor receptors include, for example, epidermal growth factorreceptor (EGFR, ErbB2 and ErbB4), platelet derived growth factorreceptor (PDGFR), vascular endothelial growth factor receptor (VEGFR),tyrosine kinase with immunoglobulin-like and epidermal growth factorhomology domains (TIE-2), insulin growth factor-I receptor (IGF-I),macrophage colony stimulating factor (cfms), BTK, ckit, cmet, fibroblastgrowth factor (FGF) receptors, Trk receptors (TrkA, TrkB, and TrkC),ephrin (Eph) receptors, and the RET protooncogene. Several inhibitors ofgrowth factor receptors are under development and include ligandantagonists, antibodies, tyrosine kinase inhibitors, anti-senseoligonucleotides and aptamers. Growth factor receptors and agents thatinhibit growth factor receptor function are described, for instance, inKath, John C., Exp. Opin. Ther. Patents (2000) 10(6):803-818; Shawver etal DDT Vol 2, No. 2 February 1997; and Lofts, F. J. et al, “GrowthFactor Receptors as Targets”, New Molecular Targets for CancerChemotherapy, Ed. Workman, Paul and Kerr, David, CRC Press 1994, London.

Tyrosine kinases, which are not growth factor receptor kinases aretermed non-receptor tyrosine kinases. Non-receptor tyrosine kinasesuseful in the present invention, which are targets or potential targetsof anti-neoplastic drugs, include cSrc, Lck, Fyn, Yes, Jak, cAbl, FAK(Focal adhesion kinase), Brutons tyrosine kinase, and Bcr-Abl. Suchnon-receptor kinases and agents which inhibit non-receptor tyrosinekinase function are described in Sinh, S, and Corey, S. J., (1999)Journal of Hematotherapy and Stem Cell Research 8 (5): 465-80; andBolen, J. B., Brugge, J. S., (1997) Annual Review of Immunology. 15:371-404.

SH2/SH3 domain blockers are agents that disrupt SH2 or SH3 domainbinding in a variety of enzymes or adaptor proteins including, PI3-K p85subunit, Src family kinases, adaptor molecules (Shc, Crk, Nck, Grb2) andRas-GAP. SH2/SH3 domains as targets for anti-cancer drugs are discussedin Smithgall, T. E. (1995), Journal of Pharmacological and ToxicologicalMethods. 34(3) 125-32.

Inhibitors of Serine/Threonine Kinases including MAP kinase cascadeblockers which include blockers of Raf kinases (Rafk), Mitogen orExtracellular Regulated Kinase (MEKs), and Extracellular RegulatedKinases (ERKs); and Protein kinase C family member blockers includingblockers of subtypes of PKCs (alpha, beta, gamma, epsilon, mu, lambda,iota, zeta), IkB kinase family (IKKa, IKKb), PKB family kinases, Aktkinase family members, and TGF beta receptor kinases. SuchSerine/Threonine kinases and inhibitors thereof are described inYamamoto, T., Taya, S., Kaibuchi, K., (1999), Journal of Biochemistry.126 (5) 799-803; Brodt, P, Samani, A., and Navab, R. (2000), BiochemicalPharmacology, 60. 1101-1107; Massague, J., Weis-Garcia, F. (1996) CancerSurveys. 27:41-64; Philip, P. A., and Harris, A. L. (1995), CancerTreatment and Research. 78: 3-27, Lackey, K. et al Bioorganic andMedicinal Chemistry Letters, (10), 2000, 223-226; and Martinez-lacaci,L., et al, Int. J. Cancer (2000), 88(1), 44-52.

Inhibitors of Phosphotidyl Inositol-3 Kinase family members includingblockers of PI3-kinase, ATM, DNA-PK, and Ku are also useful incombination with the present invention. Such kinases are discussed inAbraham, R. T. (1996), Current Opinion in Immunology. 8 (3) 412-8;Canman, C. E., Lim, D. S. (1998), Oncogene 17 (25) 3301-3308; Jackson,S. P. (1997), International Journal of Biochemistry and Cell Biology. 29(7):935-8; and Zhong, H. et al, Cancer Res, (2000) 60(6), 1541-1545.

Also useful in combination with the present invention are Myo-inositolsignaling inhibitors such as phospholipase C blockers and Myoinositolanalogues. Such signal inhibitors are described in Powis, G., andKozikowski A., (1994) New Molecular Targets for Cancer Chemotherapy ed.,Paul Workman and David Kerr, CRC Press 1994, London.

Another group of signal transduction pathway inhibitors useful incombination with the present invention are inhibitors of Ras Oncogene.Such inhibitors include inhibitors of farnesyltransferase,geranyl-geranyl transferase, and CAAX proteases as well as anti-senseoligonucleotides, ribozymes and immunotherapy. Such inhibitors have beenshown to block Ras activation in cells containing wild type mutant Ras,thereby acting as antiproliferation agents. Ras oncogene inhibition isdiscussed in Scharovsky, O. G., Rozados, V. R., Gervasoni, S. I. Matar,P. (2000), Journal of Biomedical Science. 7(4) 292-8; Ashby, M. N.(1998), Current Opinion in Lipidology. 9(2)99-102; and BioChim. Biophys.Acta, (1989) 1423(3):19-30.

As mentioned above, antibodies to receptor kinase ligand binding mayalso serve as signal transduction inhibitors. This group of signaltransduction pathway inhibitors includes the use of humanized antibodiesto the extracellular ligand binding domain of receptor tyrosine kinases.For example, Imclone C225 EGFR specific antibody (see Green, M. C. etal, Monoclonal Antibody Therapy for Solid Tumors, Cancer Treat. Rev.,(2000), 26(4), 269-286); Herceptin® ErbB2 antibody (see Tyrosine KinaseSignaling in Breast Cancer:ErbB Family Receptor Tyrosine Kinases, BreastCancer Res., 2000, 2(3), 176-183); and 2CB VEGFR2 specific antibody (seeBrekken, R. A. et al, Selective Inhibition of VEGFR2 Activity by aMonoclonal Anti-VEGF Antibody Blocks Tumor Growth in Mice, Cancer Res.(2000) 60, 5117-5124).

Receptor kinase angiogenesis inhibitors may also find use in the presentinvention. Inhibitors of angiogenesis related VEGFR and TIE2 arediscussed above in regard to signal transduction inhibitors (bothreceptors are receptor tyrosine kinases). Other inhibitors may be usedin combination with the compounds of the present invention. For example,anti-VEGF antibodies, which do not recognize VEGFR (the receptortyrosine kinase), but bind to the ligand; small molecule inhibitors ofintegrin (alpha_(v) beta₃) that will inhibit angiogenesis; endostatinand angiostatin (non-RTK) may also prove useful in combination with PLKinhibitors.

Agents used in immunotherapeutic regimens may also be useful incombination with the compounds of the invention.

Agents used in proapoptotic regimens (e.g., bcl-2 antisenseoligonucleotides) may also be used in the combination of the presentinvention. Members of the Bcl-2 family of proteins block apoptosis.Upregulation of bcl-2 has therefore been linked to chemoresistance.Studies have shown that the epidermal growth factor (EGF) stimulatesanti-apoptotic members of the bcl-2 family (i.e., mcl-1). Therefore,strategies designed to downregulate the expression of bcl-2 in tumorshave demonstrated clinical benefit and are now in Phase II/III trials,namely Genta's G3139 bcl-2 antisense oligonucleotide. Such proapoptoticstrategies using the antisense oligonucleotide strategy for bcl-2 arediscussed in Water J S et al., J. Clin. Oncol 18:1812-1823 (2000); andKitada S et al., Antisense Res. Dev. 4:71-79 (1994).

Cell cycle signaling inhibitors inhibit molecules involved in thecontrol of the cell cycle. Cyclin dependent kinases (CDKs) and theirinteraction cyclins control progression through the eukaryotic cellcycle. The coordinated activation and inactivation of differentcyclin/CDK complexes is necessary for normal progression through thecell cycle. Several inhibitors of cell cycle signaling are underdevelopment. For instance, examples of cyclin dependent kinases,including CDK2, CDK4, and CDK6 and inhibitors for the same are describedin, for instance, Rosania, et al., Exp. Opin. Ther. Patents10(2):215-230 (2000).

In one embodiment, the methods of the present invention compriseadministering to the animal a compound of the invention in combinationwith a signal transduction pathway inhibitor, particularly gefitinib(IRESSA®).

The methods and uses employing these combinations may comprise theadministration of the compound of the invention and the otherchemotherapeutic/anti-neoplastic agent either sequentially in any orderor simultaneously in separate or combined pharmaceutical compositions.When combined in the same formulation it will be appreciated that thetwo compounds must be stable and compatible with each other and theother components of the formulation and may be formulated foradministration.

When formulated separately they may be provided in any convenientformulation, in such a manner as are known for such compounds in theart. When a compound of the invention is used in combination with achemotherapeutic agent, the dose of each compound may differ from thatwhen the compound is used alone. Appropriate doses will be readilyappreciated by those skilled in the art. The appropriate dose of thecompound(s) of the invention and the other therapeutically activeagent(s) and the relative timings of administration will be selected inorder to achieve the desired combined therapeutic effect, and are withinthe expertise and discretion of the attendent clinician.

The compounds of the invention may be conveniently prepared by themethods described in the examples which follow.

The present invention also provides radiolabeled compounds of theinvention and biotinylated compounds of the invention andsolid-support-bound versions thereof. Radiolabeled compounds of theinvention and biotinylated compounds of the invention can be preparedusing conventional techniques. For example, radiolabeled compounds ofthe invention can be prepared by reacting the compound of the inventionwith tritium gas in the presence of an appropriate catalyst to produceradiolabeled compounds of the invention. In one embodiment, thecompounds are tritiated.

The radiolabeled compounds of the invention and biotinylated compoundsof the invention are useful in assays for the identification ofcompounds which inhibit PLK, for the identification of compounds for thetreatment of a condition mediated by PLK, for the treatment ofsusceptible neoplasms, for the treatment of conditions characterized byinappropriate proliferation, for the inhibition of proliferation of acell and for the inhibition of mitosis in a cell. Accordingly, thepresent invention provides an assay method for identifying suchcompounds, which method comprises the step of specifically binding theradiolabeled compound of the invention or the biotinylated compound ofthe invention to the target protein or cellular homogenates. Morespecifically, suitable assay methods will include competition bindingassays. The radiolabeled compounds of the invention and biotinylatedcompounds of the invention and solid-support-bound versions thereof, canbe employed in assays according to the methods conventional in the art.

The following examples are intended for illustration only and are notintended to limit the scope of the invention in any way, the inventionbeing defined by the claims which follow.

The following abbreviations as employed in the examples, have therecited meanings.

-   -   g gram(s)    -   mg milligram(s)    -   mol mole(s)    -   mmol millimole(s)    -   N normal    -   L liter(s)    -   mL milliliter(s)    -   μL microliter(s)    -   h hour(s)    -   min minute(s)    -   ° C. degrees Centigrade    -   HCl hydrochloric acid    -   DCM dichloromethane    -   MeOH methanol    -   EtOAc ethyl acetate    -   MgSO₄ magnesium sulfate    -   NaHCO₃ sodium bicarbonate    -   K₂CO₃ potassium carbonate    -   Na₂SO₄ sodium sulfate    -   N₂ nitrogen    -   H₂ hydrogen    -   XANTPHOS (4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene) is a        commercially available catalyst, from Aldrich.

Reagents are commercially available or are prepared according toprocedures in the literature. In the following structures, “Me” refersto the group —CH₃.

INTERMEDIATE EXAMPLE 1 Methyl5-amino-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate

Step A—Methyl5-nitro-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate

A slurry of polymer-supported triphenylphosphine (62.36 g, 2.21 mmol/g,137.8 mmol) in DCM (1.0 L) was stirred at room temperature for 10minutes. The mixture was cooled to 0° C. Methyl3-hydroxy-5-nitro-2-thiophenecarboxylate (20.00 g, 98.44 mmol), whichmay be prepared in a manner analogous to the literature procedure(Barker, J. M.; Huddleston, P. R.; Wood, M. L.; Burkitt, S. A. Journalof Chemical Research (Miniprint) 2001, 1001-1022) was added, followed by(1S)-1-[2-(trifluoromethyl)phenyl]ethanol (26.20 g, 137.8 mmol) anddi-tert-butyl azodicarboxylate (31.73 g, 137.8 mmol). The reactionmixture was stirred at room temperature for 21.25 h and then wasfiltered through a fritted funnel and concentrated. The residue wastreated with 4 N HCl in 1,4-dioxane (300 mL) and stirred at roomtemperature for 3 h. The mixture was then quenched by addition of 3 Nsodium hydroxide (300 mL) and saturated aqueous NaHCO₃ (200 mL). Themixture was extracted with DCM (3×250 mL). The combined organicfractions were dried over MgSO₄, filtered, and concentrated onto silicagel. Purification by column chromatography (0 to 25% EtOAc:hexanes)provided 36.08 g (98%) of the title compound as yellow oil. ¹H NMR (300MHz, CDCl₃): δ 7.82 (d, 1H, J=7.8 Hz), 7.68 (d, 1H, J=7.8 Hz), 7.59 (t,1H, J=7.4 Hz), 7.46 (s, 1H), 7.42 (t, 1H, J=7.6 Hz), 5.77 (q, 1H, J=6.1Hz), 3.94 (s, 3H), 1.74 (d, 3H, J=6.1 Hz).

Step B—Methyl5-amino-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate

To a flask equipped with a temperature probe, an overhead mechanicalstirrer, a reflux condenser, and an addition funnel was added ironpowder (26.84 g, 480.6 mmol) and acetic acid (130 mL). The iron/aceticacid slurry was stirred mechanically and heated to an internaltemperature of 50° C. To the addition funnel was added a solution ofmethyl5-nitro-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate(36.08 g, 96.13 mmol) in acetic acid (160 mL). The solution in theaddition funnel was then added dropwise to the iron/acetic acid slurryat a rate such that the internal temperature was maintained at <60° C.(2.5 h total addition time). The reaction mixture was cooled to roomtemperature, diluted with DCM (500 mL), and then quenched by addition of6 N sodium hydroxide (750 mL) and saturated aqueous NaHCO₃ (200 mL). Theentire mixture was then filtered through a pad of Celite to removeinsoluble material, rinsing the Celite with additional DCM (250 mL). Theaqueous and organic fractions were separated. The aqueous fraction wasextracted with EtOAc (2×400 mL). The organic fractions were combined,dried over MgSO₄, filtered, and concentrated to afford 30.66 g (92%) ofthe title compound as an orange solid. ¹H NMR (300 MHz, CDCl₃): δ 7.89(d, 1H, J=7.7 Hz), 7.62 (d, 1H, J=7.7 Hz), 7.56 (t, 1H, J=7.7 Hz), 7.36(t, 1H, J=7.7 Hz), 5.72 (s, 1H), 5.65 (q, 1H, J=6.3 Hz), 4.26 (br s,2H), 3.80 (s, 3H), 1.66 (d, 3H, J=6.3 Hz); MS (APCI): 368.00 [M+Na]⁺.

INTERMEDIATE EXAMPLE 2 Methyl5-amino-3-{[(1R)-1-(2-chlorophenyl)ethyl]-oxy}-2-thiophenecarboxylate

Step A—Methyl3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-nitro-2-thiophenecarboxylate

Methyl3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-nitro-2-thiophenecarboxylate wasprepared from methyl 3-hydroxy-5-nitro-2-thiophenecarboxylate and(1S)-1-(2-chlorophenyl)ethanol by a procedure analogous to IntermediateExample 1, Step A. ¹H NMR (400 MHz, DMSO-d₆): δ 7.96 (s, 1H), 7.65 (dd,1H, J=1.7, 7.8 Hz), 7.47 (dd, 1H, J=1.5, 7.7 Hz), 7.40 (dt, 1H, J=1.3,7.5 Hz), 7.34 (dt, 1H, J=1.9, 7.5 Hz), 5.98 (q, 1H, J=6.0 Hz), 3.85 (s,3H), 1.59 (d, 3H, J=6.2 Hz).

Step B—Methyl5-amino-3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-2-thiophenecarboxylate

Methyl5-amino-3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-2-thiophenecarboxylate wasprepared from methyl3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-nitro-2-thiophenecarboxylate bya procedure analogous to Intermediate Example 1, Step B. ¹H NMR (400MHz, DMSO-d₆): δ 7.54 (dd, 1H, J=1.8, 7.9 Hz), 7.45 (dd, 1H, J=1.4, 7.7Hz), 7.37 (dt, 1H, J=1.4, 7.7 Hz), 7.31 (dt, 1H, J=1.8, 7.6 Hz), 6.76(br s, 2H), 5.57 (q, 1H, J=6.2 Hz), 5.49 (s, 1H), 3.63 (s, 3H), 1.51 (d,3H, J=6.4 Hz); MS (ESI): 334.03 [M+Na]⁺.

EXAMPLE 15-{6-[(Methylsulfonyl)methyl]-1H-benzimidazol-1-yl}-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxamide

Route 1:

Step A—5-(Hydroxymethyl)-2-nitrophenol

To a mixture of 3-hydroxy-4-nitrobenzoic acid (5.0 g, 27.3 mmol) in1,2-dichloroethane (100 mL) was added trimethyl borate (4.9 mL, 43.7mmol), followed by boron trifluoride diethyl etherate (5.5 mL, 43.7mmol). Borane-pyridine complex (4.1 mL, 41.0 mmol) was then slowly addeddrop wise. The reaction was stirred 4 h at room temperature, then cooledto 0° C. and quenched with MeOH (10 mL). The mixture was concentratedunder vacuum and the residue taken up in toluene (200 mL), thenextracted with aqueous 1 N sodium hydroxide (3×100 mL). The combinedaqueous layers were adjusted to pH 1.0 by addition of 12 N HCl, thenextracted with EtOAc (3×250 mL). The combined organic layers were washedwith water, brine, dried over MgSO₄ and concentrated under vacuum togive 4.55 g (98%) of the title compound as a light yellow solid. ¹H NMR(400 MHz, DMSO-d₆): δ 10.87 (s, 1H), 7.85 (d, 1H, J=8.6 Hz), 7.08 (s,1H), 6.88 (dd, 1H, J=1.19, 8.51 Hz), 5.43 (s, 1H), 3.33 (s, 2H).

Step B—3-Hydroxy-4-nitrobenzyl pivalate

A mixture of 5-(hydroxymethyl)-2-nitrophenol (11.35 g, 67.15 mmol) and3-(2,2-dimethylpropanoyl)-1,3-thiazolidine-2-thione (15.0 g, 73.89mmol), which may be prepared in a manner analogous to the literatureprocedure (Yamada, S. Tetrahedron Letters 1992, 33, 2171-2174), wasstirred in toluene (670 mL) at 100° C. for 40 h, then cooled to roomtemperature. The reaction was concentrated under vacuum to a volume ofapproximately 200 mL and the resulting slurry was filtered throughfilter paper, washing the solid with cold toluene. The filtrate was thenconcentrated under vacuum and purified by silica gel chromatographyeluting with a gradient of 0-to-20% EtOAc/hexanes to give 11.09 g (65%)of the title compound as a clear yellow oil. ¹H NMR (400 MHz, DMSO-d₆):δ 11.05 (s, 1H), 7.87 (d, 1H, J=8.42 Hz), 7.06 (s, 1H), 6.90 (dd, 1H,J=1.46, 8.42 Hz), 5.09 (s, 2H), 1.18 (s, 9H).

Step C—4-Nitro-3-{[(trifluoromethyl)sulfonyl]oxy}benzyl pivalate

To a stirred, cooled (0° C.) solution of 3-hydroxy-4-nitrobenzylpivalate (11.11 g, 43.9 mmol) and N-phenyltrifluoromethanesulfonimide(16.51 g, 46.2 mmol) in DCM (220 mL) was slowly addedN,N-diisopropylethylamine (15.5 mL, 88.9 mmol). The reaction was stirredfor 45 min at 0° C., then 45 min at room temperature. The reaction wasthen concentrated under vacuum and purified by silica gel chromatographyeluting with a gradient of 5-to-20% EtOAc/hexanes to give 16.87 g (99%)of the title compound as an off white solid. ¹H NMR (400 MHz, DMSO-d₆):δ 8.36 (d, 1H, J=8.42 Hz), 7.75-7.69 (m, 2H), 5.27 (s, 2H), 1.19 (s,9H).

Step D—Methyl5-[(5-{[(2,2-dimethylpropanoyl)oxy]methyl}-2-nitrophenyl)amino]-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)thiophene-2-carboxylate

A mixture of 4-nitro-3-{[(trifluoromethyl)sulfonyl]oxy}benzyl pivalate(1.0 g, 2.60 mmol), methyl5-amino-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}-oxy)thiophene-2-carboxylate(1.34 g, 3.88 mmol), tetrakis(triphenylphosphine)palladium (0) (150 mg,0.13 mmol), triphenylphosphine (68 mg, 0.26 mmol) and K₂CO₃ (900 mg, 6.5mmol) were stirred in toluene (5.2 mL) at 100° C. for 2 h, then cooledto room temperature and filtered through Celite, washing with EtOAc andDCM. The filtrate was concentrated under vacuum and purified by silicagel chromatography eluting with a gradient of 5-to-25% EtOAc/hexane togive 1.26 g (84%) of the title compound as a red oil. ¹H NMR (400 MHz,DMSO-d₆): δ 9.75 (s, 1H), 8.09 (d, 1H, J=8.6 Hz), 7.89 (d, 1H, J=7.87Hz), 7.69-7.78 (m, 2H), 7.52 (t, 1H, J=7.59 Hz), 7.34 (s, 1H), 7.01 (dd,1H, J=1.46, 8.60 Hz), 6.62 (s, 1H), 5.70-5.75 (m, 1H), 5.07 (s, 2H),3.74 (s, 3H), 1.58 (d, 3H, J=6.22 Hz), 1.13 (s, 9H).

Step E—Methyl5-[(2-amino-5-{[(2,2-dimethylpropanoyl)oxy]methyl}phenyl)amino]-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)thiophene-2-carboxylate

A mixture of methyl5-[(5-{[(2,2-dimethylpropanoyl)oxy]methyl}-2-nitrophenyl)amino]-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)thiophene-2-carboxylate(2.42 g, 4.17 mmol) and platinum (sulfided, 5 wt % on carbon) (811 mg,0.21 mmol) in EtOAc (30 mL) was added to a high-pressure reaction flask.The reaction was purged with vacuum and N₂ gas, then H₂ gas was appliedat 50 psi for 1 h. The reaction mixture was filtered through Celite,washing with EtOAc. The filtrate was concentrated under vacuum to give2.27 g (99%) of the title compound as a tan solid. ¹H NMR (400 MHz,DMSO-d₆): δ 8.62 (s, 1H), 7.84 (d, 1H, J=7.87 Hz), 7.72 (dd, 2H, J=7.60,13.09 Hz), 7.50 (t, 1H, J=7.60 Hz), 7.01 (d, 1H, J=1.46 Hz), 6.88 (dd,1H, J=1.74, 8.15), 6.68 (d, 1H, J=8.24 Hz), 5.83 (s, 1H), 5.59-5.65 (m,1H), 4.97 (s, 2H), 4.85 (s, 2H), 3.64 (s, 3H), 1.55 (d, 3H, J=6.23 Hz),1.11 (s, 9H); MS (ESI): 551 [M+H]⁺.

Step F—Methyl5-(6-{[(2,2-dimethylpropanoyl)oxy]methyl}-1H-benzimidazol-1-yl)-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)thiophene-2-carboxylate

To a mixture of methyl5-[(2-amino-5-{[(2,2-dimethylpropanoyl)oxy]methyl}phenyl)amino]-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}-oxy)thiophene-2-carboxylate(2.27 g, 4.13 mmol) in triethylorthoformate (10 mL, 60.2 mmol) and DCM(3 mL) was added pyridinium p-toluenesulfonate (100 mg, 0.4 mmol). Thereaction was stirred at 40° C. for 1 h, then cooled to room temperature.The entire reaction mixture was loaded onto silica gel and purified bysilica gel chromatography eluting with a gradient of 0-to-50%EtOAc/hexanes to give 2.0 g (86%) of the title compound as a light tansolid. ¹H NMR (400 MHz, DMSO-d₆): δ 8.65 (s, 1H), 7.99 (d, 1H, J=7.87Hz), 7.75-7.80 (m, 2H), 7.72 (d, 1H, J=7.87 Hz), 7.63 (s, 1H), 7.53 (t,1H, J=7.60 Hz), 7.40 (s, 1H), 7.35 (d, 1H, J=8.42 Hz), 5.96 (q, 1H,J=6.10 Hz), 5.21 (s, 2H), 3.83 (s, 3H), 1.65 (d, 3H, J=6.23 Hz), 1.16(s, 9H); MS (ESI): 561 [M+H]⁺.

Step G—Methyl5-[6-(hydroxymethyl)-1H-benzimidazol-1-yl]-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)thiophene-2-carboxylate

To a stirred solution of methyl5-(6-{[(2,2-dimethylpropanoyl)oxy]methyl}-1H-benzimidazol-1-yl)-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)thiophene-2-carboxylate(5.21 g, from a different batch using procedure analogous to Example 1,Route 1, Step F, 9.30 mmol) in MeOH (24 mL) was added 0.5M sodiumhydroxide in MeOH (24.0 mL, 12 mmol). The reaction was stirred at roomtemperature for 72 h, then quenched with acetic acid (2 mL). The mixturewas diluted with DCM (350 mL) and half saturated aqueous brine solution(150 mL). The aqueous layer was extracted with DCM (250 mL). Thecombined organics were dried over MgSO₄ and concentrated under vacuum togive 4.40 g (99%) of the title compound as a light yellow solid. ¹H NMR(400 MHz, DMSO-d₆): δ 8.58 (s, 1H), 7.99 (d, 1H, J=7.87 Hz), 7.69-7.81(m, 3H), 7.51-7.58 (m, 2H), 7.38 (s, 1H), 7.30 (d, 1H, J=8.42), 5.96 (q,1H, J=6.10 Hz), 5.30 (t, 1H, J=5.77 Hz) 4.62 (d, 2H, J=5.86 Hz), 3.83(s, 3H), 1.65 (d, 3H, J=6.23 Hz); MS (ESI): 477 [M+H]⁺.

Step H—Methyl5-[6-(chloromethyl)-1H-benzimidazol-1-yl]-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)thiophene-2-carboxylate

To a stirred solution of methyl5-[6-(hydroxymethyl)-1H-benzimidazol-1-yl]-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)thiophene-2-carboxylate(1.47 g, 3.08 mmol) and triphenylphosphine (1.05 g, 4.01 mmol) in DCM(30 mL) was added N-chlorosuccinimide (0.53 g, 4.01 mmol). The reactionwas then heated to reflux and stirred for 20 minutes, then cooled toroom temperature. The reaction was diluted with DCM (400 mL) and halfsaturated aqueous brine solution (150 mL). The aqueous layer was thenextracted with DCM. The combined organic layers were dried over Na₂SO₄,concentrated under vacuum and purified by silica gel chromatographyeluting with a gradient of 10-to-60% EtOAc/hexanes to give 1.4 g (92%)of the title compound as a white solid. ¹H NMR (400 MHz, DMSO-d₆): δ8.65 (s, 1H), 7.99 (d, 1H, J=7.87 Hz), 7.72-7.81 (m, 3H), 7.69 (s, 1H),7.54 (t, 1H, J=7.69 Hz), 7.43 (d, 1H, J=8.42), 7.38 (s, 1H), 5.97 (q,1H, J=6.10 Hz), 4.91 (s, 2H), 3.84 (s, 3H), 1.66 (d, 3H, J=6.23 Hz); MS(ESI): 495 [M+H]⁺.

Step I—Methyl5-{6-[(methylthio)methyl]-1H-benzimidazol-1-yl}-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)thiophene-2-carboxylate

To a stirred mixture of methyl5-[6-(chloromethyl)-1H-benzimidazol-1-yl]-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)thiophene-2-carboxylate(200 mg, 0.40 mmol) in N,N-dimethylformamide (2.5 mL) was added sodiumthiomethoxide (37 mg, 0.52 mmol). The reaction was stirred 30 min, thendiluted with EtOAc, washed with water (5×), brine, dried over Na₂SO₄,concentrated under vacuum and purified by silica gel chromatographyeluting with a gradient of 0-to-60% EtOAc/hexanes to give 147 mg (72%)of the title compound as a white solid. MS (ESI): 507 [M+H]⁺.

StepJ—5-{6-[(Methylthio)methyl]-1H-benzimidazol-1-yl}-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)thiophene-2-carboxamide

A mixture of methyl5-{6-[(methylthio)methyl]-1H-benzimidazol-1-yl}-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)thiophene-2-carboxylate(144.0 mg, 0.28 mmol) and 7N ammonia in MeOH (18 mL, 126.0 mmol) wasadded to a high-pressure glass reaction flask. The flask was sealed,then heated to 80° C. for approx. 16 h. The flask was cooled to roomtemperature, opened, and the reaction mixture concentrated under vacuum,then purified by silica gel chromatography, eluting with a 0-to-3%gradient of MeOH/DCM with 1% ammonium hydroxide to give 130 mg (93%) ofthe title compound as a white gold solid. ¹H NMR (400 MHz, DMSO-d₆): δ8.49 (s, 1H), 7.93 (d, 1H, J=7.68 Hz), 7.85 (br s, 1H), 7.80-7.75 (m,2H), 7.69 (d, 1H, J=8.23 Hz), 7.56 (t, 1H, J=7.68 Hz), 7.39 (s, 1H),7.29 (d, 1H, J=8.42 Hz), 7.15 (brs, 1H), 7.08 (s, 1H), 5.94 (m, 1H),3.79 (s, 2H), 1.93 (s, 3H), 1.75 (d, 3H, J=6.22 Hz); MS (ESI): 492[M+H]⁺.

StepK—5-{6-[(Methylsulfonyl)methyl]-1H-benzimidazol-1-yl}-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)thiophene-2-carboxamide

To a stirred, cooled (−10° C.) solution of5-{6-[(methylthio)methyl]-1H-benzimidazol-1-yl}-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)thiophene-2-carboxamide(76 mg, 0.15 mmol) in DCM (3.0 mL) was added m-chloroperoxybenzoic acid(70 mg, 0.31 mmol). The reaction was stirred 30 min, warmed to roomtemperature and stirred 15 min, then was concentrated under vacuum. Theresidue was diluted with chloroform and washed with aqueous saturatedNaHCO₃, water, dried over Na₂SO₄, concentrated under vacuum, andpurified by silica gel chromatography eluting with a gradient of 0-to-5%MeOH/DCM, with 1% ammonium hydroxide, to give 78 mg (96%) of the titlecompound as a white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 8.57 (s, 1H),7.95 (d, 1H, J=7.87 Hz), 7.85 (br s, 1H), 7.80-7.73 (m, 3H), 7.69 (s,1H), 7.55 (t, 1H, J=7.69 Hz), 7.38 (d, 1H, J=8.42 Hz), 7.19 (s, 1H),7.12 (br s, 1H), 5.95-5.89 (m, 1H), 4.61-4.58 (m, 2H), 2.89 (s, 3H),1.75 (d, 3H, J=6.04 Hz); MS (ESI): 524 [M+H]⁺.

Route 2:

Step A—Methyl5-{6-[(methylsulfonyl)methyl]-1H-benzimidazol-1-yl}-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate

A mixture of methyl5-[6-(chloromethyl)-1H-benzimidazol-1-yl]-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)thiophene-2-carboxylate(4.53 g, from a different batch using procedure analogous to Example 1,Route 1, Step H, 9.17 mmol), methanesulfonic acid sodium salt (2.81 g,27.5 mmol) and ethanol (40.0 mL) was added to a high-pressure glassreaction flask. The flask was sealed, then heated to 85° C. forapproximately 16 h. The flask was cooled to room temperature, opened,and the reaction mixture concentrated under vacuum, then purified bysilica gel chromatography, eluting with a 5-to-35% gradient ofEtOAc/hexane to give 4.54 g (92%) of the title compound as a lightyellow solid. ¹H NMR (400 MHz, DMSO-d₆): δ 8.67 (s, 1H), 8.01 (d, 1H,J=7.87 Hz), 7.82-7.70 (m, 4H), 7.53 (t, 1H, J=7.69 Hz), 7.45 (s, 1H),7.40 (d, 1H, J=8.42 Hz), 5.95 (m, 1H), 4.63 (m, 2H), 3.83 (s, 3H), 2.90(s, 3H), 1.65 (d, 3H, J=6.204 Hz); MS (ESI): 539 [M+H]⁺.

StepB—5-{6-[(Methylsulfonyl)methyl]-1H-benzimidazol-1-yl}-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)thiophene-2-carboxamide

A mixture of methyl5-{6-[(methylsulfonyl)methyl]-1H-benzimidazol-1-yl}-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate(4.53 g, 8.42 mmol) and 7N ammonia in MeOH (250.0 mL, 1.75 mol) wasadded to a high-pressure glass reaction flask. The flask was sealed,then heated to 85° C. for approx. 36 h. The flask was cooled to roomtemperature, opened, and the reaction mixture combined with a secondbatch of methyl5-{6-[(methylsulfonyl)methyl]-1H-benzimidazol-1-yl}-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate(4.11 g, 7.63 mmol), which had also been treated with 7N ammonia in MeOH(200.0 mL, 1.40 mol) in a high-pressure glass reaction flask at 85° C.for approximately 36 h, then cooled to room temperature and opened. Thecombined reaction mixtures were concentrated under vacuum, then purifiedby silica gel chromatography, eluting with a 0-to-5% gradient ofMeOH/DCM, with 1% ammonium hydroxide, to give 7.47 g (89%) of the titlecompound as an off-white solid. MS (ESI): 524 [M+H]⁺.

EXAMPLE 23-{[(1R)-1-(2-Chlorophenyl)ethyl]oxy}-5-{6-[(methylsulfonyl)methyl]-1H-benzimidazol-1-yl}-2-thiophenecarboxamide

Step A—Methyl3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-{6-[(methylthio)methyl]-1H-benzimidazol-1-yl}thiophene-2-carboxylate

The title compound was prepared from methyl5-[6-(chloromethyl)-1H-benzimidazol-1-yl]-3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}thiophene-2-carboxylateby a procedure analogous to Example 1, Route 1, Step I. MS (ESI): 473[M+H]⁺.

StepB—3-{[(1R)-1-(2-Chlorophenyl)ethyl]oxy}-5-{6-[(methylthio)methyl]-1H-benzimidazol-1-yl}thiophene-2-carboxamide

The title compound was prepared from methyl3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-{6-[(methylthio)methyl]-1H-benzimidazol-1-yl}thiophene-2-carboxylateby a procedure analogous to Example 1, Route 1, Step J. ¹H NMR (400 MHz,DMSO-d₆): δ 8.53 (s, 1H), 7.83 (s, 1H), 7.71-7.66 (m, 2H), 7.49 (d, 1H,J=7.87 Hz), 7.45-7.35 (m, 3H), 7.29 (d, 1H, J=8.42 Hz), 7.16-7.11 (m,2H), 6.01-5.95 (m, 1H), 3.82 (s, 2H), 1.94 (s, 3H), 1.73 (d, 3H, J=6.41Hz); MS (ESI): 458 [M+H]⁺.

StepC—3-{[(1R)-1-(2-Chlorophenyl)ethyl]oxy}-5-{6-[(methylsulfonyl)methyl]-1H-benzimidazol-1-yl}-2-thiophenecarboxamide

The title compound was prepared from3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-{6-[(methylthio)methyl]-1H-benzimidazol-1-yl}thiophene-2-carboxamideby a procedure analogous to Example 1, Route 1, Step K. ¹H NMR (400 MHz,DMSO-d₆): δ 8.61 (s, 1H), 7.84 (s, 1H), 7.79 (d, 1H, J=8.24 Hz), 7.73(s, 1H), 7.70-7.67 (m, 1H), 7.49-7.47 (m, 1H), 7.44-7.33 (m, 3H), 7.26(s, 1H), 7.12 (s, 1H), 5.97-5.93 (m, 1H), 4.64-4.60 (m, 2H), 2.90 (s,3H), 1.73 (d, 3H, J=6.41 Hz); MS (ESI): 490 [M+H]⁺.

INTERMEDIATE EXAMPLE 3 Methyl5-(6-bromo-1H-benzimidazol-1-yl)-3-hydroxy-2-thiophenecarboxylate

Step A—Methyl5-(6-bromo-1H-benzimidazol-1-yl)-3-{[(1,1-dimethylethyl)(dimethyl)silyl]oxy}-2-thiophenecarboxylate

To a mixture of 5-bromo-1H-benzimidazole (43.78 g, 222.0 mmol) inchloroform (800 mL) was added N-methylimidazole (44.5 mL, 560.0 mmol),followed by methyl 2-chloro-3-oxo-2,3-dihydro-2-thiophenecarboxylate(44.8 g, 233.0 mmol). The reaction was stirred 20 h at room temperature,then N-methylimidazole (18.0 mL, 226.0 mmol) was added, followed byt-butyldimethylsilylchloride (36.8 g, 245.0 mmol). The reaction wasstirred 1 hr, then quenched with MeOH and poured into DCM and water. Theaqueous layer was extracted with DCM (3×). The combined organics werethen dried over Na₂SO₄, concentrated and chromatographed on silica gel,eluting with a 50-to-75% gradient of 25% EtOAc in hexane/hexane to give25.18 g (24%) of the title compound. MS (ESI): 467 [M+H]⁺.

Step B—Methyl5-(6-bromo-1H-benzimidazol-1-yl)-3-hydroxy-2-thiophenecarboxylate

To a stirred solution of methyl5-(6-bromo-1H-benzimidazol-1-yl)-3-{[(1,1-dimethylethyl)(dimethyl)silyl]oxy}-2-thiophenecarboxylate(25.18 g, 53.9 mmol) in THF (540.0 mL) was added 1.0M tetrabutylammoniumflouride in THF (60.0 mL, 60.0 mmol). The reaction was stirred 1.5 hthen aqueous saturated NH₄Cl was added (200 mL). The resulting slurrywas stirred 15 min then diluted with water (750 mL) and EtOAc (1.0 L).The aqueous layer was separated and its pH adjusted to 3.0 by additionof aqueous 1M HCl. The aqueous layer was then extracted with EtOAc (3×).The combined organic layers were washed with aqueous 0.1M HCl, brine,dried over MgSO₄ and concentrated under vacuum to give 19.4 g (100%) ofthe title compound as a light yellow solid. MS (ESI): 353 [M+H]⁺.

EXAMPLE 35-{6-[(4-Methylpipiperazin-1-yl)methyl]-1H-benzimidazol-1-yl}-3-{(1R)-1-[2-(trifluoromethyl)phenyl]ethoxy}thiophene-2-carboxamide

Route 1:

Step A—3-Methyl5-(6-bromo-1H-benzimidazol-1-yl)-3-{(1R)-1-[2-(trifluoromethyl)phenyl]ethoxy}thiophene-2-carboxylate

A slurry of polymer-supported triphenylphosphine (53.0 g, 2.04 mmol/g,108 mmol), (1S)-1-[2-(trifluoromethyl)phenyl]ethanol (15.4 g, 81.0mmol), and methyl5-(6-bromo-1H-benzimidazol-1-yl)-3-hydroxythiophene-2-carboxylate(Intermediate Example 3) (19.0 g, 53.9 mmol) in DCM (750 mL) was stirredat room temperature, for 10 min. The slurry was then treated withdi-tert-butyl azodicarboxylate (24.8 g, 108 mmol). The reaction mixturewas stirred for 3 h, then poured through filter paper, washing the resinsolids with DCM and MeOH. The filtrate was concentrated under vacuum andpurified by silica gel chromatography, eluting with a 5-to-50% gradientof EtOAc/hexane to give 23.8 g (84%) of the title compound as a lightyellow solid. ¹H NMR (400 MHz, DMSO-d₆): δ 8.63 (s, 1H), 7.97 (d, 1H,J=7.87 Hz), 7.80-7.71 (m, 3H), 7.65 (d, 1H, J=1.65 Hz), 7.57-7.48 (m,2H), 7.35 (s, 1H), 5.99 (q, 1H, J=5.98 Hz), 3.83 (s, 3H), 1.65 (d, 3H,J=6.04 Hz); MS (ESI): 525 [M+H]⁺.

Step B—Methyl3-{(1R)-1-[2-(trifluoromethyl)phenyl]ethoxy}-5-(6-vinyl-1H-benzimidazol-1-yl)thiophene-2-carboxylate

To a mixture of3-methyl-5-(6-bromo-1H-benzimidazol-1-yl)-3-{(1R)-1-[2-(trifluoromethyl)phenyl]ethoxy}thiophene-2-carboxylate(23.8 g, 45.4 mmol), potassium vinyltrifluoroborate (7.25 g, 54.5 mmol)and triethylamine (6.3 mL, 45.4 mmol), stirred at room temperature inn-propanol (230 mL) wasadded[1,1′-bis(diphenylphosphino)-ferrocene]dichloropalladium(II)dichloromethane complex (750 mg, 0.91 mmol). The mixture was then heatedto reflux and stirred for 3 h, then cooled to room temperature, pouredinto water and extracted with EtOAc (3×). The combined organic layerswere washed with brine, dried over MgSO₄, concentrated under vacuum andpurified by silica gel chromatography, eluting with a 10-to-40% gradientof EtOAc/hexane to give 17.06 g (80%) of the title compound as a yellowfoam solid. ¹H NMR (400 MHz, DMSO-d₆): δ 8.59 (s, 1H), 7.98 (d, 1H,J=7.87 Hz), 7.80-7.71 (m, 3H), 7.59 (s, 1H), 7.56-7.52 (m, 2H), 7.39 (s,1H), 6.85 (dd, 1H, J=10.98 and 17.75 Hz), 6.00 (q, 1H, J=6.10 Hz), 5.86(d, 1H, J=17.56 Hz), 5.31 (d, 1H, J=10.98 Hz), 3.83 (s, 3H), 1.65 (d,3H, J=6.04 Hz); MS (ESI): 473 [M+H]⁺.

Step C—Methyl5-[6-(1,2-dihydroxyethyl)-1H-benzimidazol-1-yl]-3-{(1R)-1-[2-(trifluoromethyl)phenyl]ethoxy}thiophene-2-carboxylate

To a stirred solution of methyl3-{(1R)-1-[2-(trifluoromethyl)phenyl]ethoxy}-5-(6-vinyl-1H-benzimidazol-1-yl)thiophene-2-carboxylate(17.06 g, 36.1 mmol) in 360 mL of acetone/water (3:1) was added4-methylmorpholine N-oxide (5.1 g, 43.4 mmol) followed by 2.5 weight %solution osmium tetroxide in 2-methyl-2-propanol (10.0 mL, 0.8 mmol).The reaction was stirred at room temperature for 18 h, then quenchedwith aqueous (saturated) sodium sulfite. The mixture was extracted withEtOAc (3×). The combined organic layers were washed with brine, driedover MgSO₄, concentrated under vacuum and purified by silica gelchromatography, eluting with a 1-to-8% gradient of MeOH/DCM with 1%ammonium hydroxide to give 16.72 g (92%) of the title compound as alight yellow foam solid. ¹H NMR (400 MHz, DMSO-d₆): δ 8.59 (d, 1H,J=1.46 Hz), 7.98 (d, 1H, J=7.87 Hz), 7.80-7.68 (m, 3H), 7.59-7.52 (m,2H), 7.36-7.31 (m, 2H), 5.95 (q, 1H, J=6.10 Hz), 5.37 (t, 1H, J=3.66Hz), 4.76-4.64 (m, 2H), 3.83 (s, 3H), 3.46-3.42 (m, 2H), 1.65 (d, 3H,J=6.04 Hz); MS (ESI): 507 [M+H]⁺.

Step D—Methyl5-(6-formyl-1H-benzimidazol-1-yl)-3-{(1R)-1-[2-(trifluoromethyl)phenyl]ethoxy}thiophene-2-carboxylate

To a solution of methyl5-[6-(1,2-dihydroxyethyl)-1H-benzimidazol-1-yl]-3-{(1R)-1-[2-(trifluoromethyl)phenyl]ethoxy}thiophene-2-carboxylate(16.72 g, 33.0 mmol) in 1:1:1 DCM/water/MeOH (220 mL) was added sodiumperiodate (10.58 g, 49.5 mmol). The resulting slurry was stirred for 1h, then was diluted with water and EtOAc. The aqueous layer wasextracted with EtOAc (3×). The combined organic layers were washed withbrine, dried over MgSO₄ and concentrated under vacuum to give 14.76 g(94%) of the title compound as a light yellow foam solid. ¹H NMR (400MHz, DMSO-d₆): δ10.09 (s, 1H), 8.87 (s, 1H), 8.19 (s, 1H), 8.02-7.89 (m,3H), 7.81-7.72 (m, 2H), 7.57-7.51 (m, 2H), 5.98 (q, 1H, J=6.10 Hz), 3.84(s, 3H), 1.66 (d, 3H, J=6.22 Hz); MS (ESI): 475 [M+H]⁺.

Step E—Methyl5-{6-[(4-methylpiperazin-1-yl)methyl]-1H-benzimidazol-1-yl}-3-{(1R)-1-[2-(trifluoromethyl)phenyl]ethoxy}thiophene-2-carboxylate

To a stirred solution of methyl5-(6-formyl-1H-benzimidazol-1-yl)-3-{(1R)-1-[2-(trifluoromethyl)phenyl]ethoxy}thiophene-2-carboxylate(14.76 g, 31.1 mmol), n-methylpiperazine (5.72 mL, 62.3 mmol) and aceticacid (2.1 mL, 37.4 mmol) in dichloroethane (150 mL) was added sodiumtriacetoxyborohydride (9.9 g, 46.7 mmol). The reaction was stirred for1.5 h, then aqueous 5% K₂CO₃ was added until the pH was approx. 8. Themixture was then diluted with EtOAc and water. The aqueous layer wasextracted with EtOAc (3×). The combined organic layers were washed withbrine, dried over Na₂SO₄, concentrated under vacuum and purified bysilica gel chromatography, eluting with a 1-to-8% gradient of MeOH/DCMwith 1% ammonium hydroxide to give 15.82 g (91%) of the title compoundas a light yellow foam solid. ¹H NMR (400 MHz, DMSO-d₆): δ 8.57 (s, 1H),7.98 (d, 1H, J=7.87 Hz), 7.80-7.68 (m, 3H), 7.54 (t, 1H, J=7.59 Hz),7.46 (s, 1H), 7.33-7.28 (m, 2H), 5.97 (q, 1H, J=6.16 Hz), 3.83 (s, 3H),3.55 (s, 2H), 2.45-2.20 (m, 8H), 2.13 (s, 3H), 1.66 (d, 3H, J=6.04 Hz);MS (ESI): 559 [M+H]⁺.

StepF—5-{6-[(4-Methylpiperazin-1-yl)methyl]-1H-benzimidazol-1-yl}-3-{(1R)-1-[2-(trifluoromethyl)phenyl]ethoxy}thiophene-2-carboxamide

A mixture of methyl5-{6-[(4-methylpiperazin-1-yl)methyl]-1H-benzimidazol-1-yl}-3-{(1R)-1-[2-(trifluoromethyl)phenyl]ethoxy}thiophene-2-carboxylate(15.82 g, 28.35 mmol) and 7N ammonia in MeOH (250 mL, 1.75 mol) wasadded to a high-pressure glass reaction flask. The flask was sealed,then heated to 80° C. for approx. 40 h. The flask was cooled to roomtemperature, opened, and the reaction mixture concentrated under vacuum,then purified by silica gel chromatography, eluting with a 2-to-8%gradient of MeOH/DCM with 1% ammonium hydroxide to give 14.11 g (92%) ofthe title compound as a white foam solid. ¹H NMR (400 MHz, DMSO-d₆): δ8.49 (s, 1H), 7.93 (d, 1H, J=7.87 Hz), 7.86 (br s, 1H), 7.80-7.75 (m,2H), 7.68 (d, 1H, J=8.23 Hz), 7.56 (t, 1H, J=7.68 Hz), 7.33 (s, 1H),7.28 (d, 1H, J=8.42 Hz), 7.15 (br s, 1H), 7.06 (s, 1H), 5.94 (q, 1H,J=6.10 Hz), 3.52 (s, 2H), 2.45-2.20 (m, 8H), 2.13 (s, 3H), 1.74 (d, 3H,J=6.22 Hz); MS (ESI): 544 [M+H]⁺.

Route 2:

Step A—2-bromo-4-{[(methyloxy)methyl]oxy}-1-nitrobenzene

A solution of 3-bromo-4-nitrophenol (20.0 g, 91.7 mmol) in DCM (475 mL)was stirred at 0° C. Diisopropylethylamine (19.2 mL, 110.0 mmol) wasadded, followed by the drop wise addition of a solution of chloromethylmethyl ether (7.7 mL, 100.9 mmol) in DCM (25 mL). The reaction wasstirred at 0° C. for 1 hr, then warmed to room temperature and quenchedwith water (150 mL). The mixture was poured into brine (150 mL) and theaqueous layer extracted with EtOAc (3×). The combined organic layerswere washed with brine, dried over MgSO₄, concentrated under vacuum andchromatographed on silica gel (330 g), eluting with a 0-to-25% gradientof EtOAc/hexane to give 20.0 g (83%) of the title compound as a clearorange oil. ¹H NMR (400 MHz, DMSO-d₆): δ 8.07 (d, 1H, J=8.97 Hz), 7.50(d, 1H, J=2.20 Hz), 7.21 (dd, 1H, J=8.97 and 2.38 Hz), 5.34 (s, 2H),3.39 (s, 3H).

Step B—Methyl5-[(5-{[(methyloxy)methyl]oxy}-2-nitrophenyl)amino]-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate

To a stirred solution of methyl5-amino-3-({(1R)-1-[2-(trifluoromethyl)phenyl]-ethyl}oxy)-2-thiophenecarboxylate(1.32 g, 3.82 mmol) and2-bromo-4-{[(methyloxy)methyl]oxy}-1-nitrobenzene (1.0 g, 3.82 mmol) indioxane (20 mL) was added tris(dibenzylideneacetone)dipalladium(0) (70.0mg, 0.076 mmol) and XANTPHOS (97.0 mg, 0.17 mmol) followed by cesiumcarbonate (6.2 g, 19.0 mmol). The mixture was heated to 60° C. andstirred for 12 h, then cooled to room temperature, diluted with EtOAcand filtered through Celite, washing the solids with EtOAc and DCM. Thefiltrate was concentrated under vacuum and chromatographed on silica gel(120 g), eluting with a 5-to-35% gradient of EtOAc/hexane to give 1.64 g(82%) of the title compound as a red oil. ¹H NMR (400 MHz, DMSO-d₆): δ9.85 (s, 1H), 8.12 (d, 1H, J=9.33 Hz), 7.90 (d, 1H, J=7.87 Hz), 7.74 (m,2H), 7.53 (t, 1H, J=7.68 Hz), 6.84 (d, 1H, J=2.56 Hz), 6.73-6.68 (m,2H), 5.77-5.72 (m, 1H), 5.23 (s, 2H), 3.75 (s, 3H), 3.37 (s, 3H), 1.58(d, 3H, J=6.22 Hz); MS (ESI): 527 [M+H]⁺.

Step C—Methyl5-(6-{[(methyloxy)methyl]oxy}-1H-benzimidazol-1-yl)-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate

To a high-pressure hydrogenation reaction flask was added methyl5-[(5-{[(methyloxy)methyl]oxy}-2-nitrophenyl)amino]-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate(2.0 g, 3.8 mmol), pyridinium p-toluene sulfonate (95.0 mg, 0.38 mmol),5% by weight platinum on carbon (sulfided) (740 mg, 0.19 mmol) andtrimethylorthoformate (40 mL). The flask was purged with N₂ (gas) vacuum(3×), then with H₂ (gas)/vacuum (3×). H₂ gas was then applied at 50 psifor 3 h. The reaction mixture was then filtered through Celite, washingthe solids with EtOAc and DCM. The filtrate was then concentrated undervacuum to 1.92 g (100%) of the title compound as a light yellow foamsolid. ¹H NMR (400 MHz, DMSO-d₆): δ 8.49 (s, 1H), 7.98 (d, 1H, J=8.05Hz), 7.79-7.66 (m, 3H), 7.53 (t, 1H, J=7.59 Hz), 7.35 (s, 1H), 7.22 (d,1H, J=2.20 Hz), 7.06 (dd, 1H, J=8.78 and 2.20 Hz) 5.97 (q, 1H, J=6.04Hz), 5.23 (s, 2H), 3.83 (s, 3H), 3.39 (s, 2H), 1.65 (d, 3H, J=6.22 Hz);MS (ESI): 507 [M+H]⁺.

Step D—Methyl5-(6-hydroxy-1H-benzimidazol-1-yl)-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate

To a stirred solution of methyl5-(6-{[(methyloxy)methyl]oxy}-1H-benzimidazol-1-yl)-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate(8.18 g, a different batch using procedure analogous to Example 3, Route2, Step C, 16.16 mmol) in 1:1 THF/MeOH (130 mL) was added 1N HCl inwater (65 mL, 65.0 mmol). The reaction mixture was heated to 35° C. andstirred for 72 h then cooled to room temperature. The reaction waspoured into DCM (500 mL) and water added (100 mL). The mixture wasneutralized to pH 7 by addition of aqueous (saturated) NaHCO₃. Theaqueous layer was then extracted with DCM (1×) and EtOAc (1×). Thecombined organic layers were dried over MgSO₄, concentrated undervacuum, and chromatographed on silica gel (120 g), eluting with a10-to-60% gradient of EtOAc/hexane to give 6.9 g (92%) of the titlecompound as a light salmon colored foam solid. ¹H NMR (400 MHz,DMSO-d₆): δ 9.62 (s, 1H), 8.41 (s, 1H), 8.00 (d, 1H, J=7.87 Hz),7.80-7.71 (m, 2H), 7.56-7.51 (m, 2H), 7.38 (s, 1H), 7.05 (d, 1H, J=2.01Hz), 6.81 (dd, 1H, J=8.70 and 2.11 Hz) 5.95 (m, 1H), 3.83 (s, 3H), 1.64(d, 3H, J=6.23 Hz); MS (ESI): 463 [M+H]⁺.

Step E—Methyl3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-5-(6-{[(trifluoromethyl)sulfonyl]oxy}-1H-benzimidazol-1-yl)-2-thiophenecarboxylate

To a stirred, cooled (0° C.) solution of methyl5-(6-hydroxy-1H-benzimidazol-1-yl)-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate(2.49 g, 5.38 mmol) and n-phenyltrifluoromethane sulfonamide (2.06 g,5.76 mmol) in DCM (30 mL) was added diisopropylethylamine (2.0 mL, 11.5mmol). The reaction was allowed to warm to room temperature and stirredfor 12 h. The reaction mixture was then concentrated under vacuum, andchromatographed on silica gel (120 g), eluting with a 5-to-40% gradientof EtOAc/hexane to give 3.12 g (98%) of the title compound as a lightyellow solid. ¹H NMR (400 MHz, DMSO-d₆): δ 8.76 (s, 1H), 8.01-7.94 (m,2H), 7.80-7.70 (m, 3H), 7.56-7.43 (m, 3H), 5.98 (q, 1H, J=6.10 Hz), 3.84(s, 3H), 1.65 (d, 3H, J=6.22 Hz); MS (ESI): 595 [M+H]⁺.

StepF—3-{(1R)-1-[2-(Trifluoromethyl)phenyl]ethoxy}-5-(6-vinyl-1H-benzimidazol-1-yl)thiophene-2-carboxylate

To a mixture of methyl3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-5-(6-{[(trifluoromethyl)sulfonyl]oxy}-1H-benzimidazol-1-yl)-2-thiophenecarboxylate(20.69 g, from a different batch using procedure analogous to Example 3,Route 2, Step E, 34.83 mmol), potassium vinyltrifluoroborate (5.6 g,42.10 mmol) and triethylamine (4.85 mL, 34.86 mmol), stirred at roomtemperature in n-propanol (175 mL) was added[1,1′-bis(diphenylphosphino)-ferrocene]dichloropalladium(II)dichloromethane complex (570 mg, 0.70 mmol). The mixture was then heatedto reflux and stirred for 3 h, then cooled to room temperature, pouredinto water and extracted with EtOAc (3×). The combined organic layerswere washed with brine, dried over MgSO₄, concentrated under vacuum andpurified by silica gel chromatography, eluting with a 10-to-50% gradientof EtOAc/hexane to give 12.98 g (79%) of the title compound as a lightyellow foam solid. MS (ESI): 473 [M+H]⁺.

Step G—Methyl5-[6-(1,2-dihydroxyethyl)-1H-benzimidazol-1-yl]-3-{(1R)-1-[2-(trifluoromethyl)phenyl]ethoxy}thiophene-2-carboxylate

The title compound can be prepared by a procedure analogous to Example3, Route 1, Step C.

Step H—Methyl5-(6-formyl-1H-benzimidazol-1-yl)-3-{(1R)-1-[2-(trifluoromethyl)phenyl]ethoxy}thiophene-2-carboxylate

The title compound can be prepared by a procedure analogous to Example3, Route 1, Step D.

Step I—Methyl5-{6-[(4-methylpiperazin-1-yl)methyl]-1H-benzimidazol-1-yl}-3-{(1R)-1-[2-(trifluoromethyl)phenyl]ethoxy}thiophene-2-carboxylate

The title compound can be prepared by a procedure analogous to Example3, Route 1, Step E.

StepJ—5-{6-[(4-Methylpiperazin-1-yl)methyl]-1H-benzimidazol-1-yl}-3-{(1R)-1-[2-(trifluoromethyl)phenyl]ethoxy}thiophene-2-carboxamide

The title compound can be prepared by a procedure analogous to Example3, Route 1, Step F.

Route 3:

Step A—Methyl5-{6-[(4-methylpiperazin-1-yl)methyl]-1H-benzimidazol-1-yl}-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)thiophene-2-carboxylate

To a stirred, solution of methyl5-[6-(chloromethyl)-1H-benzimidazol-1-yl]-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)thiophene-2-carboxylate(150 mg, 0.30 mmol) in dioxane (1.0 mL) was added N-methylpiperazine (50μL, 0.45 mmol). The reaction was heated at 60° C. for 18 h, cooled toroom temperature and concentrated under vacuum. The residue wasdissolved in EtOAc and water. The aqueous layer was extracted withEtOAc. The combined organic layers were washed with brine, dried oversodium sulfate, concentrated under vacuum, and purified by silica gelchromatography eluting with a gradient of 0-to-10% MeOH/DCM, with 1%ammonium hydroxide, to give 134 mg (79%) of the title compound as awhite solid. MS (ESI): 559 [M+H]⁺.

StepB—5-{6-[(4-Methylpiperazin-1-yl)methyl]-1H-benzimidazol-1-yl}-3-{(1R)-1-[2-(trifluoromethyl)phenyl]ethoxy}thiophene-2-carboxamide

The title compound can be prepared by a procedure analogous to Example3, Route 1, Step F.

Route 4:

Step A—4-[Bis(methyloxy)methyl]-2-bromo-1-nitrobenzene

A solution of 3-bromo-4-nitrobenzaldehyde (7.97 g, 34.6 mmol), which wasprepared in a manner analogous to the literature procedure (Katritzky,A. R.; Xie, L. Tetrahedron Letters 1996, 37, 347-350), trimethylorthoformate (11.4 mL, 104 mmol), and p-toluenesulfonic acid hydrate(329 mg, 1.73 mmol) in MeOH (69 mL) was refluxed for 3 h. The reactionwas then quenched by addition of saturated aqueous ammonium hydroxide (1mL) and concentrated onto silica gel. Purification by columnchromatography (10 to 25% EtOAc:hexanes) provided 8.76 g (92%) of thetitle compound as an orange oil. ¹H NMR (300 MHz, CDCl₃): δ 7.89 (m,2H), 7.59 (m, 1H), 5.47 (s, 1H), 3.38 (s, 6H).

Step B—Methyl5-({5-[bis(methyloxy)methyl]-2-nitrophenyl}amino)-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate

A solution of tris(dibenzylideneacetone) dipalladium(0) (117 mg, 0.127mmol), 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene (162 mg, 0.280mmol), methyl5-amino-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate(2.31 g, 6.69 mmol), 4-[bis(methyloxy)methyl]-2-bromo-1-nitrobenzene(1.76 g, 6.37 mmol), and cesium carbonate (10.39 g, 31.89 mmol) in1,4-dioxane (25 mL) was prepared in a round-bottom flask under N₂. Theflask was evacuated and refilled three times with N₂ and then stirred at60° C. for 16 h. The reaction mixture was then diluted withtetrahydrofuran (100 mL) and concentrated onto silica gel. Purificationby column chromatography (5 to 75% EtOAc:hexanes) provided 2.79 g (81%)of the title compound as a red foam. ¹H NMR (300 MHz, CDCl₃): δ 9.63 (brs, 1H), 8.21 (m, 1H), 7.94 (m, 1H), 7.62 (m, 2H), 7.48 (s, 1H), 7.40 (m,1H), 7.02 (m, 1H), 6.47 (s, 1H), 5.73 (q, 1H, J=6.2 Hz), 3.88 (s, 3H),3.34 (s, 1H), 3.31 (s, 3H), 3.28 (s, 3H), 1.72 (d, 3H, J=6.2 Hz); MS(ESI): 541 [M+H]⁺.

Step C—Methyl5-{6-[bis(methyloxy)methyl]-1H-benzimidazol-1-yl}-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate

To a solution of methyl5-({5-[bis(methyloxy)methyl]-2-nitrophenyl}amino)-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate(2.71 g, 5.01 mmol) in trimethyl orthoformate (50 mL) in aFischer-Porter bottle was added pyridinium p-toluenesulfonate (126 mg,0.501 mmol) and sulfided platinum on carbon (5 wt % Pt, 977 mg, 0.250mmol Pt). The mixture was hydrogenated on a Fischer-Porter hydrogenationapparatus at 50 psi of hydrogen until the uptake of hydrogen had ceased(17 h). The reaction mixture was filtered through a sintered glassfilter to remove the catalyst, washing with DCM (75 mL). The eluant wasconcentrated to afford 2.61 g (100%) of the crude title compound as anorange oil, which was carried on to the next step without purification.MS (ESI): 521 [M+H]⁺.

Step D—Methyl5-(6-formyl-1H-benzimidazol-1-yl)-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate

To a solution of crude methyl5-{6-[bis(methyloxy)methyl]-1H-benzimidazol-1-yl}-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate(2.61 g, 5.01 mmol) (from Step C, above) in acetone (20 mL) and water (5mL) was added pyridinium p-toluenesulfonate (126 mg, 0.501 mmol). Thereaction stirred for 2 h at room temperature and was then poured intowater (30 mL) and saturated aqueous NaHCO₃ (30 mL). The mixture wasextracted with DCM (2×30 mL). The combined organic fractions were driedover sodium sulfate, filtered, and concentrated onto silica gel.Purification by column chromatography (30 to 100% EtOAc:hexanes)provided 1.37 g (58%, 2 steps) of the title compound as a light yellowsolid. ¹H NMR (300 MHz, CDCl₃): δ 10.06 (s, 1H), 8.13 (s, 1H), 7.96-7.88(m, 4H), 7.72-7.61 (m, 2H), 7.44 (m, 1H), 6.82 (s, 1H), 5.84 (q, 1H,J=6.3 Hz), 3.95 (s, 3H), 1.79 (d, 3H, J=6.3 Hz); MS (ESI): 475 [M+H]⁺.

Step E—Methyl5-{6-[(4-methyl-1-piperazinyl)methyl]-1H-benzimidazol-1-yl}-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate

The title compound can be prepared by a procedure analogous to Example3, Route 1, Step E.

StepF—5-{6-[(4-Methylpiperazin-1-yl)methyl]-1H-benzimidazol-1-yl}-3-{(1R)-1-[2-(trifluoromethyl)phenyl]ethoxy}thiophene-2-carboxamide

The title compound can be prepared by a procedure analogous to Example3, Route 1, Step F.

EXAMPLE 43-{[(1R)-1-(2-Chlorophenyl)ethyl]oxy}-5-{6-[(4-methylpiperazin-1-yl)methyl]-1H-benzimidazol-1-yl}thiophene-2-carboxamide

Step A—Methyl3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-[(5-{[(2,2-dimethylpropanoyl)oxy]methyl}-2-nitrophenyl)amino]-2-thiophenecarboxylate

To a mixture of (4-nitro-3-{[(trifluoromethyl)sulfonyl]oxy}phenyl)methyl2,2-dimethylpropanoate (1.0 g, 2.59 mmol), methyl5-amino-3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-2-thiophenecarboxylate(Intermediate Example 2) (860 mg, 2.75 mmol),tris(dibenzylideneacetone)dipalladium(0) (70.0 mg, 0.076 mmol), andXANTPHOS (90.0 mg, 0.16 mmol) was added toluene (7.0 mL). Stirring wasbegun, followed by the addition of cesium carbonate (2.95 g, 9.1 mmol).The reaction was heated to 60° C. and stirred for 30 min, then cooled toroom temperature, diluted with EtOAc and filtered through Celite,washing the solids with EtOAc and DCM. The filtrate was concentratedunder vacuum and chromatographed on silica gel (40 g), eluting with a5-to-15% gradient of acetone/hexane to give 920 mg (65%) of the titlecompound as a red solid. ¹H NMR (400 MHz, DMSO-d₆): δ 9.77 (s, 1H), 8.09(d, 1H, J=8.61 Hz), 7.63 (dd, 1H, J=7.69 and 1.65 Hz), 7.46-7.30 (m,4H), 7.01 (dd, 1H, J=8.79 and 1.47 Hz), 6.67 (s, 1H), 5.76-5.70 (m, 1H),5.09 (s, 2H), 3.73 (s, 3H), 1.56 (d, 3H, J=6.23 Hz), 1.14 (s, 9H); MS(ESI): 547 [M+H]⁺.

Step B—Methyl5-[(2-amino-5-{[(2,2-dimethylpropanoyl)oxy]methyl}-phenyl)amino]-3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-2-thiophenecarboxylate

To a high-pressure hydrogenation reaction flask was added methyl3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-[(5-{[(2,2-dimethylpropanoyl)oxy]methyl}-2-nitrophenyl)amino]-2-thiophenecarboxylate(6.5 g, from a different batch using procedure analogous to Example 4,Step A, 11.9 mmol), 5% by weight platinum on carbon (sulfided) (2.2 g,0.56 mmol) and EtOAc (95 mL). The flask was purged with N₂ (gas) vacuum(3×), then with H₂ (gas) vacuum (3×). Hydrogen gas was then applied at50 psi for 3 h. The reaction mixture was then filtered through Celite,washing the solids with EtOAc and DCM. The filtrate was thenconcentrated under vacuum to give 5.46 g (89%) of the title compound asa yellow solid. MS (ESI): 517 [M+H]⁺.

Step C—Methyl3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-(6-{[(2,2-dimethylpropanoyl)oxy]methyl}-1H-benzimidazol-1-yl)-2-thiophenecarboxylate

A stirred mixture of methyl5-[(2-amino-5-{[(2,2-dimethylpropanoyl)oxy]methyl}phenyl)amino]-3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-2-thiophenecarboxylate(5.45 g, 10.5 mmol), pyridinium p-toluene sulfonate (265 mg, 1.0 mmol)and triethylorthoformate (15 mL) was heated to 40° C. for 1 h, thencooled to room temperature. The entire mixture was poured onto a silicagel cartridge (25 g) and purified by silica gel chromatography (120 g),eluting with 100% hexanes for 10 min, then a 0-to-10% EtOAc/hexanegradient to give 4.71 g (85%) of the title compound as a yellow solid.MS (ESI): 527 [M+H]⁺.

Step D—Methyl3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-[6-(hydroxymethyl)-1H-benzimidazol-1-yl]-2-thiophenecarboxylate

The title compound was prepared from methyl3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-(6-{[(2,2-dimethylpropanoyl)oxy]methyl}-1H-benzimidazol-1-yl)-2-thiophenecarboxylateby a procedure analogous to Example 1, Route 1, Step G. MS (ESI): 443[M+H]⁺.

Step E—Methyl5-[6-(chloromethyl)-1H-benzimidazol-1-yl]-3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-2-thiophenecarboxylate

The title compound was prepared from methyl3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-[6-(hydroxymethyl)-1H-benzimidazol-1-yl]-2-thiophenecarboxylateby a procedure analogous to Example 1, Route 1, Step H. MS (ESI): 461[M+H]⁺.

Step F—Methyl3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-{6-[(4-methylpiperazin-1-yl)methyl]-1H-benzimidazol-1-yl}thiophene-2-carboxylate

To a stirred mixture of methyl5-[6-(chloromethyl)-1H-benzimidazol-1-yl]-3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-2-thiophenecarboxylate(150 mg, 0.32 mmol) in dioxane (1.5 mL) was added n-methylpiperazine (55uL, 0.49 mmol) and triethylamine (136 uL, 0.97 mmol). The reaction wasthen heated at 45° C. for 18 h, cooled to room temperature andconcentrated under vacuum. The residue was dissolved in EtOAc (125 mL)and water (50 mL). The aqueous layer was then extracted with EtOAc. Thecombined organic layers were washed with brine, dried over MgSO₄,concentrated under vacuum and chromatographed on silica gel (4 g),eluting with a 0-to-10% gradient of MeOH/DCM, with 1% ammoniumhydroxide, to give 123 mg (72%) of the title compound as a light yellowsolid. MS (ESI): 525 [M+H]⁺.

StepG—3-{[(1R)-1-(2-Chlorophenyl)ethyl]oxy}-5-{6-[(4-methylpiperazin-1-yl)methyl]-1H-benzimidazol-1-yl}thiophene-2-carboxamide

The title compound was prepared from methyl3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-{6-[(4-methylpiperazin-1-yl)methyl]-1H-benzimidazol-1-yl}thiophene-2-carboxylateby a procedure analogous to Example 3, Route 1, Step F. ¹H NMR (400 MHz,DMSO-d₆): δ 8.52 (s, 1H), 7.83 (s, 1H), 7.69 (d, 2H, J=8.24 Hz),7.51-7.34 (m, 4H), 7.28 (d, 1H, J=8.24 Hz), 7.16-7.11 (m, 2H), 5.98 (q,1H, J=6.35 Hz), 3.55 (s, 2H), 2.42-2.22 (m, 8H), 2.13 (s, 3H), 1.72 (d,3H, J=6.23 Hz); MS (ESI): 510 [M+H]⁺.

INTERMEDIATE EXAMPLE 4 Methyl3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-(6-hydroxy-1H-benzimidazol-1-yl)-2-thiophenecarboxylate

Step A—2-Bromo-4-({[4-(methyloxy)phenyl]methyl}oxy)-1-nitrobenzene

2-Bromo-4-fluoro-1-nitrobenzene (20.0 g, 90.9 mmol) and 4-methoxybenzylalcohol (22.7 mL, 182 mmol) were dissolved in DCM (400 mL) withstirring. 1N sodium hydroxide solution (400 mL) was added followed bytetrabutylammonium hydrogensulfate (3.09 g, 9.10 mmol). The reaction wasstirred for 8 h and poured into a separatory funnel. The layers wereseparated and the aqueous was extracted once with DCM and once withdiethyl ether. The combined organic layers were dried over MgSO₄,filtered, and concentrated in vacuo. Purification by flashchromatography afforded 28.01 g (91%) of the title compound. ¹H NMR (400MHz, CDCl₃): δ 8.03 (d, 1H, J=9.2 Hz), 7.50 (d, 1H, J=2.6 Hz), 7.39-7.34(m, 2H), 7.17 (dd, 1H, J=2.7, 9.0 Hz), 6.95-6.91 (m, 2H), 5.14 (s, 2H),3.73 (s, 3H).

Step B—Methyl3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-{[5-({[4-(methyloxy)pheny]methyl}oxy)-2-nitrophenyl]amino}-2-thiophenecarboxylate

2-Bromo-4-({[4-(methyloxy)phenyl]methyl}oxy)-1-nitrobenzene (20.19 g,59.7 mmol) and methyl5-amino-3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-2-thiophenecarboxylate(18.60 g, 59.7 mmol) were dissolved in 1,4-dioxane (500 mL) withstirring in a flask equipped with a mechanical stirrer, refluxcondenser, and thermometer. The solution was degassed for 75 min bybubbling nitrogen through the stirring solution.9,9-Dimethyl-4,5-bis(diphenylphosphino)xanthene (1.52 g, 2.63 mmol),cesium carbonate (97.26 g, 299 mmol), and tris(dibenzylideneacetone)dipalladium(0) (1.09 g, 1.19 mmol) were added. The reaction was heatedto 60° C. and stirred for 16 h. The reaction was cooled to roomtemperature and filtered through Celite. The solid was washed with 20%MeOH in DCM. The filtrate was concentrated onto approximately 200 g ofsilica gel. The solid was placed in a fritted funnel and washed with 10%EtOAc in DCM. The filtrate was concentrated in vacuo. Purification byflash chromatography provided 27.18 g (80%) of the title compound. ¹HNMR (400 MHz, CDCl₃): δ 9.87 (s, 1H), 8.10 (d, 1H, J=9.5 Hz), 7.63 (m,1H), 7.39-7.29 (m, 4H), 7.23 (m, 1H), 6.96-6.90 (m, 2H), 6.80 (d, 1H,J=2.6 Hz), 6.75 (s, 1H), 6.69 (dd, 1H, J=2.6, 9.3 Hz), 5.75 (q, 1H,J=6.3 Hz), 5.03 (AB, 2H, J_(AB)=13.2 Hz, J_(AB)=11.3 Hz), 3.74 (s, 3H),3.74 (s, 3H), 1.55 (d, 3H, J=6.4 Hz).

Step C—Methyl5-{[2-amino-5-({[4-(methyloxy)phenyl]methyl}oxy)-phenyl]amino}-3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-2-thiophenecarboxylate

Methyl3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-{[5-({[4-(methyloxy)phenyl]-methyl}oxy)-2-nitrophenyl]amino}-2-thiophenecarboxylate(27.18 g, 47.8 mmol) was dissolved in EtOAc (400 mL) with stirring.Sulfided platinum (5% weight on carbon, 2.80 g) was added, and thereaction was placed under 1 atm of H₂ using a balloon apparatus. After36 h an additional amount of catalyst (2.80 g) was added and stirringwas continued under 1 atm of hydrogen. After 16 h more, the reaction wasfiltered through a Celite pad washing with EtOAc. The filtrate wasconcentrated to afford the title compound, which was immediately carriedinto the next step. ¹H NMR (400 MHz, CDCl₃): δ 8.66 (br s, 1H), 7.56(dd, 1H, J=1.7, 7.8 Hz), 7.41-7.09 (m, 5H), 6.93-6.88 (m, 2H), 6.71 (d,1H, J=2.8 Hz), 6.65 (d, 1H, J=8.6 Hz), 6.57 (dd, 1H, J=2.7, 8.6 Hz),5.87 (s, 1H), 5.62 (q, 1H, J=6.4 Hz), 4.82 (s, 2H), 4.46 (br s, 2H),3.73 (s, 3H), 3.64 (s, 3H), 1.52 (d, 3H, J=6.2 Hz).

Step D—Methyl3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-[6-({[4-(methyloxy)phenyl]methyl}oxy)-1H-benzimidazol-1-yl]-2-thiophenecarboxylate

Methyl5-{[2-amino-5-({[4-(methyloxy)phenyl]methyl}oxy)phenyl]amino}-3-{[(1R)-1-(2-chlorophenyl)-ethyl]oxy}-2-thiophenecarboxylatewas dissolved in trimethyl orthoformate (100 mL) and diethyl ether (100mL) with stirring. Pyridinium p-toluenesulfonate (0.601 g, 2.39 mmol)was added in a single portion. The reaction was stirred for 2.5 h andquenched by the addition of triethylamine (approximately 3 mL). Themixture was concentrated and purified by flash chromatography to afford25.45 g (97% over 2 steps) of the title compound. ¹H NMR (400 MHz,CDCl₃): δ 8.47 (s, 1H), 7.71 (dd, 1H, J=1.6, 8.2 Hz), 7.63 (d, 1H, J=9.0Hz), 7.43-7.08 (m, 7H), 7.00 (dd, 1H, J=2.4, 8.8 Hz), 6.96-6.90 (m, 2H),5.97 (q, 1H, J=6.4 Hz), 5.03 (AB, 2H, J_(AB)=17.1 Hz, J_(AB)=11.3 Hz),3.80 (s, 3H), 3.73 (s, 3H), 1.60 (d, 3H, J=6.4 Hz).

Step E—Methyl3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-(6-hydroxy-1H-benzimidazol-1-yl)-2-thiophenecarboxylate

Methyl3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-[6-({[4-(methyloxy)phenyl]-methyl}oxy)-1H-benzimidazol-1-yl]-2-thiophenecarboxylate(25.45 g, 46.4 mmol) was dissolved in DCM (120 mL) and cooled to 0° C.with stirring. Trifluoroacetic acid (40.0 mL, 519 mmol) was addeddropwise via addition funnel. The reaction was stirred for 1 h andsodium hydroxide (20.0 g, 500 mmol) in water (120 mL) was added dropwisevia addition funnel. The pH of the mixture was then adjusted to neutralwith saturated NaHCO₃ solution. The reaction was poured into aseparatory funnel, and the layers were separated. The aqueous layer waswashed with EtOAc. The combined organic layers were dried over MgSO₄,filtered, and concentrated in vacuo. Purification by flashchromatography afforded 14.86 g (75%) of the title compound. ¹H NMR (400MHz, CDCl₃): δ 9.62 (s, 1H), 8.45 (s, 1H), 7.74 (dd, 1H, J=1.7, 7.7 Hz),7.53 (d, 1H, J=8.8 Hz), 7.46-7.38 (m, 3H), 7.32 (m, 1H), 7.08 (d, 1H,J=2.2 Hz), 6.79 (dd, 1H, J=2.2, 8.6 Hz), 5.94 (q, 1H, J=6.2 Hz), 3.79(s, 3H), 1.60 (d, 3H, J=6.2 Hz).

INTERMEDIATE EXAMPLE 5 Methyl5-(6-hydroxy-1H-benzimidazol-1-yl)-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate

Step A—Methyl5-{[5-({[4-(methyloxy)phenyl]methyl}oxy)-2-nitrophenyl]amino}-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate

The title compound was prepared from methyl5-amino-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate and2-bromo-4-({[4-(methyloxy)phenyl]methyl}oxy)-1-nitrobenzene by aprocedure analogous to Intermediate Example 4, Step B. MS (ESI): 603[M+H]⁺.

Step B—Methyl5-{[2-amino-5-({[4-(methyloxy)phenyl]methyl}oxy)phenyl]amino}-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate

The title compound was prepared from methyl5-{[5-({[4-(methyloxy)phenyl]methyl}oxy)-2-nitrophenyl]amino}-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylateby a procedure analogous to Intermediate Example 4, Step C. MS (ESI):573 [M+H]⁺.

Step C—Methyl5-(6-hydroxy-1H-benzimidazol-1-yl)-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate

Methyl5-{[2-amino-5-({[4-(methyloxy)phenyl]methyl}oxy)phenyl]amino}-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate(11 g, 19.19 mmol) was dissolved in 100 mL of trimethyl orthoformatewith stirring. Pyridinium p-toluenesulfonate (0.502 g, 1.91 mmol) wasadded in a single portion. The reaction was stirred for 2.5 h. Themixture was concentrated and the crude methyl5-[6-({[4-(methyloxy)phenyl]methyl}oxy)-1H-benzimidazol-1-yl]-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylatewas dissolved in chloroform (75 mL) and cooled to 0° C. with stirring.Trifluoroacetic acid (50.0 mL, 649 mmol) was added. The reaction wasstirred for 1 h and allowed to come to room temperature. The mixture wasconcentrated while cooling to remove most of the trifluoroacetic acid.The mixture was dissolved in chloroform (200 mL). The reaction waspoured into a separatory funnel, and the layers were separated. The pHof the mixture was then adjusted to neutral with saturated NaHCO₃solution. The aqueous layer was washed with chloroform. The combinedorganic layers were dried over MgSO₄, filtered, and concentrated invacuo. Purification by flash chromatography afforded 8.16 g (92% over 2steps) of the title compound. ¹H NMR (400 MHz, CDCl₃): δ 7.88 (d, 1H,J=7.87 Hz), 7.83 (s, 1H), 7.66-7.55 (m, 3H), 7.40 (t, 1H, J=7.7 Hz),6.92 (d, 1H, J=2.2 Hz), 6.85 (dd, 1H, J=2.3, 8.7 Hz), 5.78 (q, 1H,J=6.23 Hz), 5.47 (s, 1H), 3.91 (s, 3H), 1.75 (d, 3H, J=6.23 Hz); MS(ESI): 463 [M+H]⁺.

EXAMPLE 55-[6-(4-Piperidinyloxy)-1H-benzimidazol-1-yl]-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxamide

Step A—1,1-Dimethylethyl4-({1-[5-[(methyloxy)carbonyl]-4-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thienyl]-1H-benzimidazol-6-yl}oxy)-1-piperidinecarboxylate

Methyl5-(6-hydroxy-1H-benzimidazol-1-yl)-3-({(1R)-1-[2-(trifluoromethyl)phenyl]-ethyl}oxy)-2-thiophenecarboxylate(0.478 g, 1.03 mmol), cesium carbonate (0.470 g, 1.44 mmol), and4-(Toluene-4-sulfonyloxy)-piperidine-1-carboxylic acid tert-butyl ester(0.439 g, 1.24 mmol) were combined in 10 mL of N,N-dimethylformamide andheated to 60° C. with stirring. The reaction was heated for 36 h andcooled to room temperature. The mixture was poured into EtOAc and water,and the layers were separated. The organic layer was washed with brine,and the combined aqueous layers were extracted with EtOAc. The combinedorganic layers were dried over MgSO₄, filtered, and concentrated invacuo. Purification by flash chromatography afforded 0.482 g (72%) ofthe title compound. ¹H NMR (400 MHz, DMSO-d₆) δ 8.43 (s, 1H), 7.95 (dd,J=7.7 Hz, 1H), 7.78-7.66 (m, 2H), 7.63 (d, J=8.8 Hz, 1H), 7.50 (m, 1H),7.29 (s, 1H), 7.09 (d, J=2.2 Hz, 1H), 7.01 (dd, J=8.8, 2.2 Hz, 1H), 5.97(q, J=6.2 Hz, 1H), 4.59 (m, 1H), 3.81 (s, 3H), 3.65-3.56 (m, 2H),3.25-3.15 (m, 2H), 1.91-1.82 (m, 2H), 1.63 (d, J=6.2 Hz, 3H), 1.59-1.49(m, 2H), 1.38 (s, 9H). MS m/z 646 (M+1).

Step B—Methyl5-[6-(4-piperidinyloxy)-1H-benzimidazol-1-yl]-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate

1,1-Dimethylethyl4-({1-[5-[(methyloxy)carbonyl]-4-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thienyl]-1H-benzimidazol-6-yl}oxy)-1-piperidinecarboxylate(1.84 g, from a different batch using procedure analogous to Example 5,Step A, 2.85 mmol) was dissolved in 30 mL of DCM with stirring andcooled to 0° C. Trifluoroacetic acid (10.0 mL, 130 mmol) was addeddropwise via addition funnel. The reaction was stirred for 1 h, and 2 Nsodium hydroxide solution (60 mL) was added dropwise via additionfunnel. Saturated aqueous NaHCO₃ solution was used to adjust the pH tobasic. The mixture was poured into a separatory funnel, and the layerswere separated. The aqueous layer was washed once with DCM and once withdiethyl ether. The combined organic layers were dried over MgSO₄,filtered, and concentrated in vacuo. Purification by flashchromatography provided 1.37 g (88%) of the title compound. ¹H NMR (400MHz, DMSO-d₆): δ 8.42 (s, 1H), 7.95 (d, J=7.9 Hz, 1H), 7.78-7.67 (m,2H), 7.61 (d, J=8.6 Hz, 1H), 7.61 (m, 1H), 7.30 (s, 1H), 7.05 (d, J=2.2Hz, 1H), 6.97 (dd, J=8.8, 2.2 Hz, 1H), 5.96 (q, J=6.1 Hz, 1H), 4.41 (m,1H), 3.80 (s, 3H), 2.97-2.88 (m, 2H), 2.59-2.49 (m, 2H), 1.92-1.83 (m,2H), 1.63 (d, J=6.1 Hz, 3H), 1.51-1.38 (m, 2H). MS m/z 546 (M+1).

StepC—5-[6-(4-Piperidinyloxy)-1H-benzimidazol-1-yl]-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxamide

Methyl5-[6-(4-piperidinyloxy)-1H-benzimidazol-1-yl]-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate(0.154 g, 0.282 mmol) was dissolved in 7 N ammonia in MeOH (12.0 mL,84.0 mmol) in a sealed tube and heated to 80° C. for 2 days. Thereaction was cooled to room temperature and concentrated in vacuo.Purification by flash chromatography afforded 0.129 g (86%) of the titlecompound. ¹H NMR (400 MHz, DMSO-d₆) δ 8.34 (s, 1H), 7.91 (d, J=8.0 Hz,1H), 7.81 (br s, 1H), 7.78-7.70 (m, 2H), 7.61 (d, J=8.4 Hz, 1H), 7.53(m, 1H), 7.12 (br s, 1H), 7.03 (s, 1H), 7.00-6.93 (m, 2H), 5.94 (q,J=6.2 Hz, 1H), 4.44 (m, 1H), 3.03-2.94 (m, 2H), 2.70-2.61 (m, 2H),1.96-1.86 (m, 2H), 1.72 (d, J=6.2 Hz, 3H), 1.59-1.46 (m, 2H). MS m/z 531(M+1).

EXAMPLE 63-{[(1R)-1-(2-Chlorophenyl)ethyl]oxy}-5-[6-(4-piperidinyloxy)-1H-benzimidazol-1-yl]-2-thiophenecarboxamide

Step A—1,1-Dimethylethyl4-[(1-{4-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-[(methyloxy)carbonyl]-2-thienyl}-1H-benzimidazol-6-yl)oxy]-1-piperidinecarboxylate

Methyl3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-(6-hydroxy-1H-benzimidazol-1-yl)-2-thiophenecarboxylate(2.00 g, 4.66 mmol), triphenylphosphine (4.89 g, 18.6 mmol), and t-butyl4-hydroxy-1-piperidinecarboxylate (1.88 g, 9.34 mmol) were dissolved inDCM (50 mL) with stirring and cooled to 10° C. Diisopropylazodicarboxylate (1.84 mL, 9.35 mmol) was added dropwise via syringe.The reaction was stirred for 5 min and allowed to warm to roomtemperature. The reaction was stirred for 4 h and adsorbed onto silicagel. Purification by flash chromatography afforded the title compoundalong with small amounts of impurity. ¹H NMR (400 MHz, DMSO-d₆): δ 8.47(s, 1H), 7.70 (dd, 1H, J=1.6, 7.7 Hz), 7.64 (d, 1H, J=8.8 Hz), 7.44-7.37(m, 2H), 7.34 (s, 1H), 7.31 (m, 1H), 7.15 (d, 1H, J=2.4 Hz), 7.01 (dd,1H, J=2.2, 8.8 Hz), 5.97 (q, 1H, J=6.2 Hz), 4.59 (m, 1H), 3.80 (s, 3H),3.65-3.56 (m, 2H), 3.27-3.14 (m, 2H), 1.92-1.81 (m, 2H), 1.60 (d, 3H,J=6.2 Hz), 1.60-1.47 (m, 2H), 1.38 (s, 9H); MS (ESI): 612 [M+H]⁺.

Step B—Methyl3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-[6-(4-piperidinyloxy)-1H-benzimidazol-1-yl]-2-thiophenecarboxylate

1,1-Dimethylethyl4-[(1-{4-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-[(methyloxy)-carbonyl]-2-thienyl}-1H-benzimidazol-6-yl)oxy]-1-piperidinecarboxylatewas dissolved in DCM (60 mL) and cooled to 0° C. Trifluoroacetic acid(15.0 mL, 195 mmol) was added dropwise via addition funnel. The reactionwas stirred for 1.5 h, and 2 N sodium hydroxide solution (88 mL) wasadded dropwise via addition funnel. Saturated aqueous NaHCO₃ solutionwas used to adjust the pH to ˜8. The mixture was poured into aseparatory funnel, and the layers were separated. The aqueous layer waswashed with DCM (3×) and EtOAc (1×). The combined organic layers weredried over MgSO₄, filtered, and concentrated in vacuo. Purification byflash chromatography provided 1.95 g (82% over 2 steps) of the titlecompound. ¹H NMR (400 MHz, DMSO-d₆): δ 8.46 (s, 1H), 7.71 (dd, 1H,J=1.7, 7.7 Hz), 7.62 (d, 1H, J=8.8 Hz), 7.46-7.38 (m, 2H), 7.35 (s, 1H),7.32 (m, 1H), 7.09 (d, 1H, J=2.2 Hz), 6.97 (dd, 1H, J=2.2, 8.8 Hz), 5.97(q, 1H, J=6.2 Hz), 4.42 (m, 1H), 3.80 (s, 3H), 2.96-2.88 (m, 2H),2.58-2.49 (m, 2H), 1.93-1.84 (m, 2H), 1.60 (d, 3H, J=6.2 Hz), 1.51-1.39(m, 2H); MS (ESI): 512 [M+1]⁺.

StepC—3-{[(1R)-1-(2-Chlorophenyl)ethyl]oxy}-5-[6-(4-piperidinyloxy)-1H-benzimidazol-1-yl]-2-thiophenecarboxamide

Methyl3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-[6-(4-piperidinyloxy)-1H-benzimidazol-1-yl]-2-thiophenecarboxylate(0.150 g, 0.293 mmol) was dissolved in 7 N ammonia in MeOH (12.0 mL,84.0 mmol) in a sealed tube and heated to 80° C. for 48 h. The solutionwas concentrated down and recharged with fresh 7 N ammonia in MeOH (12.0mL, 84.0 mmol) and heated to 110° C. for 72 h. The reaction was cooledto room temperature and concentrated in vacuo. Purification by flashchromatography afforded 0.126 g (87%) of the title compound. ¹H NMR (400MHz, DMSO-d₆): δ 8.38 (s, 1H), 7.79 (br s, 1H), 7.66 (dd, 1H, J=1.6, 7.7Hz, 1H), 7.61 (d, 1H, J=8.8 Hz), 7.45 (dd, 1H, J=1.3, 7.8 Hz), 7.40 (m,1H), 7.84 (m, 1H), 7.11 (s, 1H), 7.11 (br s, 1H), 7.01 (d, 1H, J=2.2Hz), 6.96 (dd, 1H, J=2.3, 8.7 Hz), 5.98 (q, 1H, J=6.2 Hz), 4.41 (m, 1H),2.98-2.89 (m, 2H), 2.62-2.53 (m, 2H), 1.94-1.84 (m, 2H), 1.70 (d, 3H,J=6.2 Hz), 1.54-1.40 (m, 2H); MS (ESI): 497 [M+H]⁺.

EXAMPLE 75-{6-[(1-Methyl-4-piperidinyl)oxy]-1H-benzimidazol-1-yl}-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxamide

Step A—Methyl5-{6-[(1-methyl-4-piperidinyl)oxy]-1H-benzimidazol-1-yl}-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate

Methyl5-[6-(4-piperidinyloxy)-1H-benzimidazol-1-yl]-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate(0.200 g, 0.367 mmol) was dissolved in DCM (4 mL) and MeOH (2 mL).Acetic acid (0.025 mL, 0.44 mmol) and formaldeldehyde (0.055 mL, 37% inwater, 0.74 mmol) were added via syringe. Sodium triacetoxyborohydride(0.117 g, 0.552 mmol) was added in a single portion. The reaction wasstirred for 1 h and quenched with saturated NaHCO₃ solution. The mixturewas poured into DCM and half-saturated aqueous NaHCO₃. The layers wereseparated, and the aqueous layer was washed with DCM (3×) and EtOAc(1×). The combined organic layers were dried over MgSO₄, filtered, andconcentrated in vacuo. Purification by flash chromatography afforded0.150 g (73%) of the title compound. ¹H NMR (400 MHz, DMSO-d₆): δ 8.43(s, 1H), 7.96 (d, J=7.7 Hz, 1H), 7.78-7.68 (m, 2H), 7.62 (d, J=9.0 Hz,1H), 7.51 (m, 1H), 7.32 (s, 1H), 7.06 (br s, 1H), 6.98 (m, 1H), 5.97 (q,J=6.0 Hz, 1H), 4.41 (m, 1H), 3.80 (s, 3H), 3.26 (s, 3H), 2.52-2.43 (m,2H), 2.27-2.11 (m, 2H), 1.98-1.85 (m, 2H), 1.73-1.60 (m, 2H), 1.62 (d,J=6.0 Hz, 3H). MS (ESI): 560 [M+H]⁺.

StepB—5-{6-[(1-Methyl-4-piperidinyl)oxy]-1H-benzimidazol-1-yl}-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxamide

Methyl5-{6-[(1-methyl-4-piperidinyl)oxy]-1H-benzimidazol-1-yl}-3-({(1R)-1-[2-(trifluoromethyl)phenyl]ethyl}oxy)-2-thiophenecarboxylate(0.148 g, 0.264 mmol) was dissolved in 7 N ammonia in MeOH (12.0 mL,84.0 mmol) in a sealed tube and heated to 80° C. for 24 hours. Thereaction was cooled to room temperature and concentrated in vacuo.Purification by flash chromatography afforded 0.138 g (96%) of the titlecompound. ¹H NMR (400 MHz, DMSO-d₆): δ 8.35 (s, 1H), 7.92 (d, J=7.7 Hz,1H), 7.82 (br s, 1H), 7.80-7.71 (m, 2H), 7.64-7.51 (m, 2H), 7.12 (br s,1H), 7.06 (s, 1H), 6.99-6.94 (m, 2H), 5.95 (q, J=6.2 Hz, 1H), 4.36 (m,1H), 2.64-2.53 (m, 2H), 2.23-2.11 (m, 2H), 2.17 (s, 3H), 1.94-1.84 (m,2H), 1.73 (d, J=6.2 Hz, 3H), 1.71-1.57 (m, 2H). MS (ESI): 545 [M+H]⁺.

EXAMPLE 83-{[(1R)-1-(2-Chlorophenyl)ethyl]oxy}-5-{6-[(1-methyl-4-piperidinyl)oxy]-1H-benzimidazol-1-yl}-2-thiophenecarboxamide

Step A—Methyl3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-{6-[(1-methyl-4-piperidinyl)oxy]-1H-benzimidazol-1-yl}-2-thiophenecarboxylate

Methyl3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-[6-(4-piperidinyloxy)-1H-benzimidazol-1-yl]-2-thiophenecarboxylate(0.260 g, 0.508 mmol) was dissolved in DCM (4 mL) and MeOH (2 mL).Acetic acid (0.035 mL, 0.61 mmol) and formaldeldehyde (0.076 mL, 37% inwater, 1.0 mmol) were added via syringe. Sodium triacetoxyborohydride(0.161 g, 0.760 mmol) was added in a single portion. The reaction wasstirred for 2 h and poured into DCM and half-saturated aqueous NaHCO₃.The layers were separated, and the aqueous layer was washed with DCM andEtOAc. The combined organic layers were dried over MgSO₄, filtered, andconcentrated in vacuo. Purification by flash chromatography afforded0.215 g (80%) of the title compound. ¹H NMR (400 MHz, DMSO-d₆): δ 8.48(s, 1H), 7.73 (dd, 1H, J=1.8, 7.7 Hz), 7.63 (d, 1H, J=8.8 Hz), 7.47-7.40(m, 2H), 7.38 (s, 1H), 7.34 (m, 1H), 7.12 (d, 1H, J=2.2 Hz), 6.99 (dd,1H, J=2.2, 8.8 Hz), 5.98 (q, 1H, J=6.2 Hz), 4.41 (m, 1H), 3.80 (s, 3H),2.65-2.54 (m, 2H), 2.24-2.13 (m, 2H), 2.17 (s, 3H), 1.97-1.87 (m, 2H),1.72-1.61 (m, 2H), 1.62 (d, 3H, J=6.2 Hz); MS (ESI): 526 [M+H]⁺.

StepB—3-{[(1R)-1-(2-Chlorophenyl)ethyl]oxy}-5-{6-[(1-methyl-4-piperidinyl)oxy]-1H-benzimidazol-1-yl}-2-thiophenecarboxamide

Methyl3-{[(1R)-1-(2-chlorophenyl)ethyl]oxy}-5-{6-[(1-methyl-4-piperidinyl)oxy]-1H-benzimidazol-1-yl}-2-thiophenecarboxylate(0.214 g, 0.407 mmol) was dissolved in 7 N ammonia in MeOH (12.0 mL,84.0 mmol) in a sealed tube and heated to 80° C. for 2.5 days. Thereaction was cooled to room temperature and concentrated in vacuo.Purification by flash chromatography afforded 0.208 g (100%) of thetitle compound. ¹H NMR (400 MHz, DMSO-d₆): δ 8.39 (s, 1H), 7.80 (br s,1H), 7.67 (dd, 1H, J=1.5, 7.6 Hz), 7.62 (d, 1H, J=8.6 Hz), 7.45 (m, 1H),7.41 (m, 1H), 7.34 (m, 1H), 7.13 (s, 1H), 7.11 (brs, 1H), 7.02 (d, 1H,J=2.0 Hz), 6.97 (dd, 1H, J=2.2, 8.8 Hz), 5.99 (q, 1H, J=6.2 Hz), 4.37(m, 1H), 2.63-2.53 (m, 2H), 2.22-2.14 (m, 2H), 2.17 (s, 3H), 1.95-1.86(m, 2H), 1.71 (d, 3H, J=6.2 Hz), 1.70-1.59 (m, 2H); MS (ESI): 511[M+H]⁺.

COMPARATIVE EXAMPLES

Comparative Example numbers 121, 126, 127, 136 and 143 can be preparedusing methods known in the art, including those described in PCTPublication No. WO2004/014899 to SmithKline Beecham Corp.

Num- ber Structure 121

126

127

136

143

BIOLOGICAL EXAMPLES

I. Assay for Inhibition of PLK1

A. Preparation of 6×N-terminal His-tagged PLK kinase domain 6×N-terminalHis-tagged PLK kinase domain (amino acids 21-346 proceeded byMKKGHHHHHHD) SEQ ID: No. 1. was prepared from baculovirus infected T. nicells under polyhedrin promoter control. All procedures were performedat 4° C. Cells were lysed in 50 mM HEPES, 200 mM NaCl, 50 mM imidazole,5% glycerol; pH 7.5. The homogenate was centrifuged at 14K rpm in aSLA-1500 rotor for 1 h and the supernatant filtered through a 1.2 micronfilter. The supernatant was loaded onto a Nickel chelating Sepharose(Amersham Pharmacia) column and washed with lysis buffer. Protein waseluted using 20%, 30% and 100% buffer B steps where buffer B is 50 mMHEPES, 200 mM NaCl, 300 mM imidazole, 5% glycerol; pH 7.5. Fractionscontaining PLK were determined by SDS-PAGE. Fractions containing PLKwere diluted five-fold with 50 mM HEPES, 1 mM DTT, 5% glycerol; pH 7.5,then loaded on an SP Sepharose (Amersham Pharmacia) column. Afterwashing the column with 50 mM HEPES, 1 mM DTT, 5% glycerol; pH 7.5, PLKwas step eluted with 50 mM HEPES, 1 mM DTT, 500 mM NaCl; 5% glycerol; pH7.5. PLK was concentrated using a 10 kDa molecular weight cutoffmembrane and then loaded onto a Superdex 200 gel filtration (AmershamPharmacia) column equilibrated in 25 mM HEPES, 1 mM DTT, 500 mM NaCl, 5%glycerol; pH 7.5. Fractions containing PLK were determined by SDS-PAGE.PLK was pooled, aliquoted and stored at −80° C. Samples were qualitycontrolled using mass spectrometry, N-terminal sequencing and amino acidanalysis.

B. Enzyme activity +/− inhibitors was determined as follows: Allmeasurements were obtained under conditions where signal productionincreased linearly with time and enzyme. Test compounds were added towhite 384-well assay plates (0.1 μL for 10 μL and some 20 μL assays, 1μL for some 20 μL assays) at variable known concentrations in 100% DMSO.DMSO (1-5% final, as appropriate) and EDTA (65 mM in reaction) were usedas controls. Reaction Mix was prepared as follows at 22° C.:

-   -   25 mM HEPES, pH 7.2    -   15 mM MgCl2    -   1 μM ATP    -   0.05 μCi/well ³³P-_(γ) ATP (10 Ci/mMol)    -   1 μM substrate peptide (Biotin-Ahx-SFNDTLDFD) SEQ ID:No. 2.    -   0.15 mg/mL BSA    -   1 mM DTT    -   2 nM PLK1 kinase domain (added last)

Reaction Mix (10 or 20 μL) was quickly added to each well immediatelyfollowing addition of enzyme via automated liquid handlers and incubated1-1.5 h at 22° C. The 20 μL enzymatic reactions were stopped with 50 μLof stop mix (50 mM EDTA, 4.0 mg/ml Streptavidin SPA beads in StandardDulbecco's PBS (without Mg²⁺ and Ca²⁺), 50 μM ATP) per well. The 10 μLreactions were stopped with 10 μL of stop mix (50 mM EDTA, 3.0 mg/mLStreptavidin-coupled SPA Imaging Beads (“LeadSeeker”) in StandardDulbecco's PBS (without Mg²⁺ and Ca²⁺), 50 μM ATP) per well. Plates weresealed with clear plastic seals, spun at 500×g for 1 min or settledovernight, and counted in Packard TopCount for 30 seconds/well (regularSPA) or imaged using a Viewlux imager (LeadSeeker SPA). Signal abovebackground (EDTA controls) was converted to percent inhibition relativeto that obtained in control (DMSO-only) wells.

C. Results

The data are reported in Table 1 below. In Table 1, +=pIC₅₀<6; ++=pIC₅₀6-8; +++=pIC₅₀>8.

II. Methylene Blue Growth Inhibition Assay—Inhibition of CellProliferation by PLK1 Inhibitors

Generally, exponentially growing cell lines of different tumor origins,cultured in appropriate media containing 10% fetal bovine serum at 37°C. in a 5% CO₂ incubator were plated at low density (less than 2000cells/well) in 96-well plates. Twenty four hours post-plating, cellswere treated with different concentrations of test compounds rangingfrom 10 uM to 0.04 nM. Several wells were left untreated as a control.Seventy two hours post-treatment, cell numbers were determined using 100μl per well of methylene blue (Sigma M9140) (0.5% in 50:50Ethanol:water). Stain was incubated at room temperature for 30 minutesbefore plates were rinsed and dye solubilized in 1% N-lauroyl sarcosine,sodium salt, (Sigma L5125, in PBS) (further details of a methylene blueassay are described below). Plates were read on a microplate reader,measuring the OD at 620 nm.

Percent inhibition of cell growth was expressed as percent proliferationrelative to 100% proliferation (control). Concentration of test compoundthat inhibited 50% of cell growth (IC₅₀) was determined by 4 parameterfit of data using XLfit, (value of no cell control was substracted fromall samples for background). The data are shown in Table 1 below andrepresent a compilation of several different experiments, each performedusing the general parameters outlined above, although minor variationsmay have been employed in some instances.

In one assay, normal human foreskin fibroblasts (HFF), human colon(HCT116, RKO), lung (H460, A549) and breast (MCF7) tumor cell lines werecultured in high glucose DMEM (Life Technologies) containing 10% fetalbovine serum (FBS) at 37° C. in a humidified 5% CO₂, 95% air incubator.Cells were harvested using trypsin/EDTA, counted using a haemocytometer,and plated in 100 μL of culture media per well, at the followingdensities, in a 96-well tissue culture plate (Falcon 3075): HFF 5,000cells/well, HCT116 3,000 cells/well, RKO 2,500 cells/well, H460 2,500cells/well, A549 5,000 cells/well, MCF7 4,000 cells/well. The next day,compounds were diluted in low glucose DMEM containing 100 μg/mLgentamicin, at twice the final required concentration, from 10 mM stocksolutions in DMSO. 100 μL/well of these dilutions were added to the 100μL of media currently in the assay plates. Medium containing 0.6% DMSOwas added to control wells. The final concentration of DMSO in all wellswas 0.3%. Cells were incubated at 37° C., 5% CO₂ for 72 h. Medium wasremoved by aspiration. Cell biomass was estimated by staining cells with80 μL per well methylene blue (Sigma M9140, 0.5% in 50:50ethanol:water), and incubation at room temperature for 30-60 min. Stainwas removed by aspiration and the plates rinsed by immersion in water,then air-dried. To release stain from the cells, 100 μL ofsolubilization solution was added (1% N-lauroyl sarcosine, Sodium salt,Sigma L5125, in PBS), and plates were shaken gently for about 30 min.Optical density at 620 nM was measured on a microplate reader. Percentinhibition of cell growth was calculated relative to vehicle treatedcontrol wells. Concentration of compound that inhibits 50% of cellgrowth (IC₅₀) was interpolated using nonlinear regression(Levenberg-Marquardt) and the equation, y=V_(max)*(1−(x/(K+x)))+Y2,where “K” was equal to the IC₅₀.

The data are reported in Table 1 below. In Table 1, +=IC₅₀>1 μM; ++=IC₅₀0.1-1 μM: +++=IC₅₀<0.1 μM.

III. Determination of Protein Binding to Human Plasma Proteins UsingEquilibrium Dialysis

96 Well Plate (High Throughput Dialysis): Stock solutions of compoundswere spiked into human plasma at a target concentration of 2000 ng/mL.The mixture was inverted gently several times to insure homogeneity andtriplicate 50 μL aliquots were collected to verify initialconcentrations. Following assembly of dialysis plate (HTDialysismembrane strips, molecular weight cut off limit of 12,000-14,000daltons), spiked plasma (150 μL) was placed in the donor compartment ofthe well and Phosphate Buffered Saline pH 7.4 (150 μL) in the receivercompartment. Eight wells were set up per compound and plasma type. Platewas placed in a 37° C. incubator on a plate shaker. Following the 6 hincubation period, the plate was removed. Single 50 μL aliquots fromeach donor and receiver compartment (per well) were analyzed. Sampleanalysis was by LC/MS/MS (results reported as Drug Peak Area/InternalStandard Peak Area ratios). Protein binding assay can also be performedusing dialysis cells instead of HT Dialysis 96 well plates. The data arereported in Table 1 below. In Table 1, % Protein Binding, +=>98%;++=95-98%: +++=<95%.

IV. High Throughput Solubility Assay

Two samples are prepared for each compound. One (the standard sample)contains the compound at a fixed concentration of 20 μM in anaqueous/organic mixed solvent cocktail. The other (the test sample)contains the compound at a maximum concentration of 200 μM in pH 7.4,0.05M phosphate buffer and shaking for 24 h. The test sample is filteredby 0.45μ filter and then spun for 10 min to remove any undissolvedsolid. HPLC analyses are preformed on these samples. The peak areas areused for computing solubility. The data are reported in Table 1 below.In Table 1, solubility, +=<30 μM; ++=30-100 μM: +++=>100 μM.

TABLE 1 HCT116 H460 MCF7 A549 RKO HFF % PLK1 IC₅₀ IC₅₀ IC₅₀ IC₅₀ IC₅₀IC₅₀ Protein Solubility Example # pIC₅₀ (μM) (μM) (μM) (μM) (μM) (μM)Binding (μM) 1 +++ +++ +++ +++ +++ +++ ++ ++ ++ 2 +++ +++ +++ +++ ++++++ ++ ++ ++ 3 +++ +++ +++ +++ +++ +++ +++ +++ +++ 4 +++ +++ +++ +++ ++++++ +++ ND +++ 5 +++ +++ +++ +++ +++ +++ ++ ++ +++ 6 +++ +++ +++ ND ++++++ ++ +++ +++ 7 +++ +++ +++ +++ +++ +++ ++ +++ +++ 8 +++ +++ +++ ND ++++++ +++ +++ +++ Com. +++ ++ +++ ++ +++ +++ + + + Ex 127 Com. +++ ++++++ + +++ +++ ++ + ND Ex 126 Com. ++ + + + + + + ++ ND Ex 121 Com. ++ ++++ ++ ND ++ + +++ ND Ex 136 Com. ++ ++ + ++ + ++ + +++ + Ex 143 ND: NotDetermined

Table 1 shows that the instantly claimed compounds possess superiorproperties over the comparative examples tested. For example, examplecompounds 1-8 have superior enzyme and cell potency over comparativeexamples 121, 136 and 143 in PLK1 enzyme assay and methylene blue cellproliferation assay in multiple cell lines examined. Example compounds1-8 have superior solubility in pH 7.4, 0.05 M phosphate buffer overcomparative example 127. Example compounds 1-3 and 5-8 have superiorprotein binding in human serum by equilibrium dialysis assay overcomparative example 126 and 127.

V. Cell-Titer-Glo—Inhibition of Cell Proliferation by PLK1 Inhibitors

Exponentially growing cell lines of different tumor origins, cultured inappropriate media containing 10% fetal bovine serum at 37° C. in a 5%CO₂ incubator were plated at low density (less than 2000 cells/well) in96-well plates. Twenty four hours post-plating, cells were treated withdifferent concentrations of test compounds ranging from 10 uM to 0.04nM. Several wells were left untreated as a control. Seventy two hourspost-treatment, cell numbers were determined using 50-100 ul per well ofCellTiter-Glo (Promega #G7573). Plates were incubated at roomtemperature for 15 minutes and the chemiluminescent signal was read onthe Victor V or Envison 2100 reader.

Percent inhibition of cell growth was expressed as percent proliferationrelative to 100% proliferation (control). Concentration of test compoundthat inhibited 50% of cell growth (IC₅₀) was determined by 4 parameterfit of data using XLfit, (value of no cell control was substracted fromall samples for background). The Cell-Titer Glo data are shown in Table2 below and represent a compilation of several different experiments,each performed using the general parameters outlined above, althoughminor variations may have been employed in some instances. In Table 2,+=IC₅₀>1 μM; ++=IC₅₀ 0.5-1 μM: +++=IC₅₀<0.5 μM.

TABLE 2 Cell Line Ex 1 Ex 2 Ex 3 Ex 4 Ex 5 Ex 6 Ex 7 Ex 8 HCT116 IC₅₀ NDND +++ ND +++ ND ND ND A549-L IC₅₀ ND ND +++ ND +++ ND ND ND COLO205IC₅₀ ND ND +++ ND +++ ND ND ND HT29 IC₅₀ ND ND +++ ND +++ ND ND ND MX-1IC₅₀ ND ND +++ ND +++ ND ND ND SKOV-3 IC₅₀ ND ND +++ ND +++ ND ND NDLNCaP IC₅₀ ND ND +++ ND ND ND ND ND P388 IC₅₀ ND ND +++ ND ND ND ND NDH1299 IC₅₀ ND ND +++ ND +++ ND ND ND Hela IC₅₀ ND ND +++ ND ND ND ND NDHN5 IC₅₀ ND ND +++ ND ND ND ND ND MCF7 IC₅₀ ND ND +++ ND +++ ND ND NDMV522 IC₅₀ ND ND +++ ND +++ ND ND ND MDA-MB-468 IC₅₀ ND ND +++ ND +++ NDND ND PANC-1 IC₅₀ ND ND +++ ND ND ND ND ND MiaPaca IC₅₀ ND ND +++ ND NDND ND ND ASPC3 IC₅₀ ND ND +++ ND ND ND ND ND BXPC3 IC₅₀ ND ND +++ ND NDND ND ND

1. A compound selected from:

wherein * indicates a chiral carbon; and a pharmaceutically acceptablesalt thereof.
 2. A compound according to claim 1, wherein thestereochemistry of the chiral carbon is R.
 3. A compound according toclaim 1, comprising greater than 50% by weight of an enantiomer selectedfrom formula E-1, F-1, G-1 and H1:


4. A pharmaceutical composition comprising a compound according to claim1 and a pharmaceutically acceptable carrier, diluent or excipient. 5.The pharmaceutical composition according to claim 4 further comprising achemotherapeutic agent.
 6. An enantiomerically pure compound selectedfrom formula E-1, F-1, G-1 and H1:

and a pharmaceutically acceptable salt thereof.
 7. A pharmaceuticalcomposition comprising a compound according to claim 6.